Altering microbial populations &amp; modifying microbiota

ABSTRACT

The invention relates to methods, uses, systems, arrays, engineered nucleotide sequences and vectors for inhibiting bacterial population growth or for altering the relative ratio of sub-populations of first and second bacteria in a mixed population of bacteria. The invention is particularly useful, for example, for treatment of microbes such as for environmental, medical, food and beverage use. The invention relates inter alia to methods of controlling microbiologically influenced corrosion (MIC) or biofouling of a substrate or fluid in an industrial or domestic system.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part of co-pending U.S.application Ser. No. 15/460,962 filed 16 Mar. 2017, which is aContinuation of co-pending U.S. application Ser. No. 15/160,405 filedMay 20, 2016 which is a Continuation application under 35 U.S.C. §120 ofInternational Patent Application No. PCT/EP2016/059803 filed on May 3,2016, which claims priority to GB Application Numbers 1507773.8, Filedon May 6, 2015; 1507774.6, Filed on May 6, 2015; 1507775.3, Filed on May6, 2015; 1507776.1, Filed on May 6, 2015; 1508461.9, Filed on May 17,2015; 1509366.9, Filed on May 31, 2015; 1510891.3, Filed on Jun. 20,2015; 1518402.1, Filed on Oct. 17, 2015; 1600417.8, Filed on Jan. 10,2016; and 1600418.6, Filed on Jan. 10, 2016, the contents of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to methods of inhibiting bacterial populationgrowth, altering the relative ratio of sub-populations of first andsecond bacteria in a mixed population of bacteria, nucleic acid arraysfor this purpose and vectors comprising the arrays. The inventionrelates to engineered systems for modifying host cell nucleic acid,components of such systems and application of these in industry andmedicine. The invention is particularly useful, for example, fortreatment of microbes such as for environmental, food and beverage use.The invention relates inter alia to methods of controllingmicrobiologically influenced corrosion (MIC) or biofouling of asubstrate or fluid in an industrial or domestic system. The inventionalso relates to treated fluids and vectors for use in the methods. Inembodiments, the methods use horizontal transfer of arrays. Theinvention also provides arrays comprised by mobile genetic elements(MGEs) for this purpose and vectors comprising these arrays.

BACKGROUND OF THE INVENTION

Inhibiting bacterial population growth and altering the relative ratiosof different bacterial species in a mixture finds application in a widerange of industries and settings, for example for treatment ofwaterways, drinking water or in other environmental settings.Application is also found in altering bacteria in humans and non-humananimals, eg, livestock, for reducing pathogenic infections or forre-balancing gut or oral microbiota. Recently, there has been interestin analysing the relative proportions of gut bacteria in humans withdiffering body mass or obesity profiles, or in investigating possiblebacterial influence in disease contexts such as Crohn's disease.

Although bacterial innate immune mechanisms against phage abound, anextensively documented bacterial adaptive immune system is theCRISPR/Cas system. Engineered CRISPR/Cas systems have been used forprecise modification of nucleic acid in various types of prokaryotic andeukaryotic cells, ranging from bacterial to animal and plant cells (eg,see Jiang W et al (2013)). Prokaryotes, such as bacteria and archaea,encode adaptive immune systems, called CRISPR/Cas (clustered regularlyinterspaced short palindromic repeats/CRISPR associated), to provideresistance against mobile invaders, such as viruses (eg, bacteriophage)and plasmids. Reference is made to Seed et al (2013), which explainsthat bacteriophages (or phages) are the most abundant biologicalentities on earth, and are estimated to outnumber their bacterial preyby tenfold. The constant threat of phage predation has led to theevolution of a broad range of bacterial immunity mechanisms that in turnresult in the evolution of diverse phage immune evasion strategies,leading to a dynamic co-evolutionary arms race.

Host immunity is based on incorporation of invader DNA sequences in amemory locus (CRISPR array), the formation of guide RNAs from thislocus, and the degradation of cognate invader DNA (protospacer) situatedadjacent a protospacer adjacent motif (PAM). See, for exampleWO2010/075424. The host CRISPR array comprises various elements: aleader (including a promoter) immediately 5′ of one or morerepeat-spacer-repeat units where the repeats are identical and thespacers differ. By acquiring spacer sequence from invading virus orplasmid nucleic acid, the host defense system is able to incorporate newspacers into the CRISPR array (each spacer flanked by repeats) to act asa memory to tackle future invasion by the virus or plasmid. It has beenobserved that recently-acquired spacers tend to be inserted into thehost array directly after the leader.

Reference is made to Heler et al (2014), which explains that CRISPR lociand their associated genes (Cas) confer bacteria and archaea withadaptive immunity against phages and other invading genetic elements. Afundamental requirement of any immune system is the ability to build amemory of past infections in order to deal more efficiently withrecurrent infections. The adaptive feature of CRISPR-Cas immune systemsrelies on their ability to memorize DNA sequences of invading moleculesand integrate them in between the repetitive sequences of the CRISPRarray in the form of ‘spacers’. The transcription of a spacer generatesa small antisense RNA that is used by RNA-guided Cas nucleases to cleavethe invading nucleic acid in order to protect the cell from infection.The acquisition of new spacers allows the CRISPR-Cas immune system torapidly adapt against new threats and is therefore termed ‘adaptation’(ie, vector sequence spacer acquisition).

Seed et al (2013) reported a remarkable turn of events, in which aphage-encoded CRISPR/Cas system was used to counteract a phageinhibitory chromosomal island of the bacterial host. A successful lyticinfection by the phage reportedly was dependent on sequence identitybetween CRISPR spacers and the target chromosomal island. In the absenceof such targeting, the phage-encoded CRISPR/Cas system could acquire newspacers to evolve rapidly and ensure effective targeting of thechromosomal island to restore phage replication. Bondy-Denomy et al(2012) describe the early observed examples of genes that mediate theinhibition of a CRISPR/Cas system. Five distinct ‘anti-CRISPR’ geneswere found in the genomes of bacteriophages infecting Pseudomonasaeruginosa. Mutation of the anti-CRISPR gene of a phage rendered itunable to infect bacteria with a functional CRISPR/Cas system, and theaddition of the same gene to the genome of a CRISPR/Cas-targeted phageallowed it to evade the CRISPR/Cas system.

Immature RNAs are transcribed from CRISPR arrays and are subsequentlymatured to form crRNAs. Some CRISPR/Cas systems also comprise sequencesencoding trans-activating RNAs (tracrRNAs) that are able to hybridise torepeats in the immature crRNAs to form pre-crRNAs, whereby furtherprocessing produces mature, or crRNAs. The architecture of cRNAs variesaccording to the type (Type I, II or III) CRISPR/Cas system involved.

CRISPR-associated (cas) genes are often associated with CRISPR arrays.Extensive comparative genomics have identified many different cas genes;an initial analysis of 40 bacterial and archaeal genomes suggested thatthere may be 45 cas gene families, with only two genes, cas1 and cas2,universally present. Cas1 and Cas2 are believed to be essential for newspacer acquisition into arrays, thus are important in mechanisms ofdeveloping resistance to invader nucleic acid from phage or plasmids.Nuñez et al (2015) reportedly demonstrated the Cas1-Cas2 complex to bethe minimal machinery that catalyses spacer DNA acquisition andapparently explain the significance of CRISPR repeats in providingsequence and structural specificity for Cas1-Cas2-mediated adaptiveimmunity.

CRISPR/Cas systems also include sequences expressing nucleases (eg,Cas9) for cutting invader nucleic acid adjacent cognate recognitionmotifs (PAMs) in invader nucleotide sequences. PAM recognition ofnucleases is specific to each type of Cas nuclease. The PAMs in theinvader sequences may lie immediately 3′ of a protospacer sequence, withnucleases typically cutting 3-4 nucleotides upstream of (5′ of) the PAM.The conservation of the PAM sequence differs between CRISPR-Cas systemsand appears to be evolutionarily linked to cas 1 and the leadersequence. Fineran et al (2014) observed that Invaders can escape typeI-E CRISPR-Cas immunity in Escherichia coli K12 by making pointmutations in a region (the “seed region”) of the protospacer or itsadjacent PAM, but hosts quickly restore immunity by integrating newspacers in a positive-feedback process involving acquisition(“priming”). To date, the PAM has been well characterized in a number oftype I and type II systems and the effect of mutations in theprotospacer has been documented (see references 5, 14, 23, 46, 47 inFineran et al (2014)). Fineran et al (2014) concluded that their resultsdemonstrated the critical role of the PAM and the seed sequence, inagreement with previous work.

Semenova et al (2011) investigated the role of the seed sequence andconcluded that that in the case of Escherichia coli subtype CRISPR/Cassystem, the requirements for crRNA matching are strict for the seedregion immediately following the PAM. They observed that mutations inthe seed region abolish CRISPR/Cas mediated immunity by reducing thebinding affinity of the crRNA-guided Cascade complex to protospacer DNA.

The stages of CRISPR immunity for each of the three major types ofadaptive immunity are as follows:—

(1) Acquisition begins by recognition of invading DNA by Cas1 and Cas2and cleavage of a protospacer;

(2) A protospacer sequence is ligated to the direct repeat adjacent tothe leader sequence; and

(3) Single strand extension repairs the CRISPR and duplicates the directrepeat.

The crRNA processing and interference stages occur differently in eachof the three major types of CRISPR systems. The primary CRISPRtranscript is cleaved by Cas to produce crRNAs. In type I systemsCas6e/Cas6f cleave at the junction of ssRNA and dsRNA formed by hairpinloops in the direct repeat. Type II systems use a trans-activating(tracr) RNA to form dsRNA, which is cleaved by Cas9 and RNaseIII. TypeIII systems use a Cas6 homolog that does not require hairpin loops inthe direct repeat for cleavage. In type II and type III systemssecondary trimming is performed at either the 5′ or 3′ end to producemature crRNAs. Mature crRNAs associate with Cas proteins to forminterference complexes. In type I and type II systems, base-pairingbetween the crRNA and the PAM causes degradation of invading DNA. TypeIII systems do not require a PAM for successful degradation and in typeIII-A systems base-pairing occurs between the crRNA and mRNA rather thanthe DNA, targeted by type III-B systems.

STATEMENTS OF INVENTION

First Configuration of the Invention

The inventors believe that they have demonstrated for the first timeinhibition of population growth of a specific bacterial strain in amixed consortium of bacteria that naturally occur together in microbiota(human, animal or environmental microbiota) with one or more of thefollowing features:—

Population growth inhibition by

-   -   targeting wild-type cells;    -   harnessing of wild-type endogenous Cas nuclease activity;    -   targeting essential and antibiotic resistance genes;    -   wherein the targets are wild-type sequences.

The inventors have demonstrated this in a mixed bacterial populationwith the following features:—

-   -   targeting bacterial growth inhibition in a mixed population of        human microbiota (such as gut microbiota) species;    -   wherein the population comprises three different species;    -   comprising selective killing of one of those species and sparing        cells of the other species;    -   targeting cell growth inhibition in the presence of a        phylogenetically-close other species, which is spared such        inhibition;    -   targeting cell growth inhibition in a mixed population        comprising target Firmicutes species and non-firmicutes species;    -   targeting cell growth inhibition of a specific Firmicutes strain        whilst sparing a different Firmicutes species in a mixed        population;    -   targeting cell growth inhibition of a specific gram positive        bacterial strain whilst sparing a different gram positive        bacterial species in a mixed population;    -   targeting a pathogenic (in humans) bacterial species whilst        sparing a commensul human gut bacterial species;    -   targeting a pathogenic bacterial species whilst sparing a        probiotic human gut bacterial species;    -   targeting cell growth inhibition in a mixed bacterial population        on a surface;    -   achieving at least a 10-fold growth inhibition of a specific        bacterial species alone or when mixed with a plurality of other        bacterial species in a consortium; and    -   achieving at least a 10-fold growth inhibition of two different        strains of a specific bacterial species.

The ability to harness endogenous Cas activity in wild-type cells isvery useful for in situ treatment of host cell infections in organisms(humans and animals, for example) and the environment. Treatment ofwild-type (ie, non-engineered or pre-manipulated) bacterial populations,such as human, animal or plant microbiota can also be addressed usingthe invention. The ability to effect selective growth inhibition in amixed population is useful for addressing bacterial populations, such ashuman, animal or plant microbiota, or for addressing environmentalmicrobiomes. This feature is also useful for producing medicaments (eg,bacterial cell transplants for administration to a human or animalsubject for any treatment or prevention disclosed herein; or forproducing a herbicide or insecticide composition comprising the productbacterial population of the invention), wherein the selective killingcan be used to selectively alter the ratio of different bacteria in amixed population to produce an altered bacterial population which is themedicament, herbicide or insecticide; or from which the medicament,herbicide or insecticide is produced. For example, the medicament can beintranasally transplanted into a human or animal recipient to effectsuch treatment or prevention.

In the worked Example below, growth inhibition was addressed in abacterial population (a gram positive Firmicutes population) on a solidsurface. A >10-fold population growth inhibition was achieved. Targetingwas directed to an antibiotic resistance gene. The invention will beuseful in inhibiting the growth of antibiotic-resistant bacteria,wherein the target sequence is a sequence of an antibiotic resistancegene. In an example, co-administration of the engineered nucleotidesequence with the antibiotic may be effective. This may provide morecomplete treatment or prevention of host cell infection in human oranimal subjects and/or enable the reduction of therapeutically-effectiveantibiotic dose for administration to a human or animal. This is usefulin view of the increasing worry regarding over-administration ofantibiotics and the development of resistance in human and animalpopulations. The invention also finds application ex vivo and in vitrofor treating an industrial or medical fluid, surface, apparatus orcontainer (eg, for food, consumer goods, cosmetics, personal healthcareproduct, petroleum or oil production); or for treating a waterway,water, a beverage, a foodstuff or a cosmetic, wherein the host cell(s)are comprised by or on the fluid, surface, apparatus, container,waterway, water, beverage, foodstuff or cosmetic. The invention findsapplication also in control of corrosion, biofilms and biofouling. Thefirst configuration thus provides the following concepts:—

Use of a host modifying (HM) CRISPR/Cas system for altering the relativeratio of sub-populations of first and second bacteria in a mixedpopulation of bacteria, the second bacteria comprising host cells,

for each host cell the system comprising components according to (i) to(iv):—(i) at least one nucleic acid sequence encoding a Cas nuclease;(ii) a host cell target sequence and an engineered host modifying (HM)CRISPR array comprising a spacer sequence (HM-spacer) and repeatsencoding a HM-crRNA, the HM-crRNA comprising a sequence that hybridisesto the host cell target sequence to guide said Cas to the target in thehost cell to modify the target sequence;(iii) an optional tracrRNA sequence or a DNA sequence expressing atracrRNA sequence;(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that transforms the host cell,whereby the HM-crRNA guides Cas to the target to modify the hostCRISPR/Cas system in the host cell; andwherein the target sequence is modified by the Cas whereby the host cellis killed or host cell growth is reduced.

A host modifying (HM) CRISPR/Cas system for the use of claim 1 formodifying a target nucleotide sequence of a bacterial host cell, thesystem comprising components according to (i) to (iv):—

(i) at least one nucleic acid sequence encoding a Cas nuclease;(ii) a host cell target sequence and an engineered host modifying (HM)CRISPR array comprising a spacer sequence (HM-spacer) and repeatsencoding a HM-crRNA, the HM-crRNA comprising a sequence that is capableof hybridising to the host target sequence to guide said Cas to thetarget in the host cell to modify the target sequence;(iii) an optional tracrRNA sequence or a DNA sequence for expressing atracrRNA sequence;(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that can transform the hostcell, whereby the HM-crRNA guides Cas to the target to modify the hostCRISPR/Cas system in the host cell.

This is exemplified by the worked Examples herein where we showselective host cell growth inhibition by at least 10-fold in a mixed andnon-mixed cell population. The mixture simulates a combination ofspecies and strains found in human microbiota.

Use of wild-type endogenous Cas nuclease activity of a bacterial hostcell population to inhibit growth of the population, wherein each hostcell has an endogenous CRISPR/Cas system having wild-type Cas nucleaseactivity, the use comprising transforming host cells of the population,wherein each transformed host cell is transformed with an engineerednucleotide sequence for providing host modifying (HM) cRNA or guide RNA(gRNA) in the host cell, the HM-cRNA or gRNA comprising a sequence thatis capable of hybridising to a host cell target protospacer sequence forguiding endogenous Cas to the target, wherein the cRNA or gRNA iscognate to an endogenous Cas nuclease of the host cell that has saidwild-type nuclease activity and following transformation of the hostcells growth of the population is inhibited.

Use (optionally the use is according to the use of the immediatelypreceding paragraph above) of a host modifying (HM) CRISPR/Cas systemfor killing or reducing the growth of bacterial host cells, for eachhost cell the system comprising components according to (i) to (iv):—

(i) at least one nucleic acid sequence encoding a Cas nuclease;(ii) an engineered host modifying (HM) CRISPR array comprising a spacersequence (HM-spacer) and repeats encoding a HM-crRNA, the HM-crRNAcomprising a sequence that hybridises to a host cell target sequence toguide said Cas to the target in the host cell to modify the targetsequence;(iii) an optional tracrRNA sequence or a DNA sequence expressing atracrRNA sequence;(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that transforms the host cell,whereby the HM-crRNA guides Cas to the target to modify the targetsequence in the host cell;Wherein the Cas nuclease is endogenous to the host cell; and wherein thetarget sequence is modified by the Cas whereby the host cell is killedor host cell growth is reduced.

Thus, the HM-cRNA is capable of hybridising to the host cell targetsequence to guide said Cas to the target in the host cell to modify thetarget sequence.

In an alternative, HM-crRNA and tracrRNA are comprised by a single guideRNA (gRNA).

By harnessing endogenous Cas nuclease, embodiments of the invention useendogenous Cas nuclease activity (ie, without the need for prior geneticmodification of the host cell to activate or enhance the nucleaseactivity). Thus, in an example, the Cas nuclease is encoded by awild-type gene of the host cell. In an example, the nuclease is activeto achieve the cell killing or growth inhibition without inactivation ofan endogenous Cas nuclease (or Cas nuclease gene) repressor in the hostcell. Thus, the invention can address wild-type bacterial populationswithout the need for prior manipulation to bring about effectiveCas-mediated cell killing or growth reduction. Thus, the population canbe exposed to the cRNA when the population is in its wild-typeenvironment (such as a waterway or comprised by a human or animalmicrobiome).

In an example, the first bacteria are Bacteroidetes (eg, Bacteroides)cells. In an example, the second bacteria are Firmicutes cells. Themethod is, for example, used to alter the ratios in a gut microbiotapopulation (eg, ex vivo or in vivo), which is for example for treatingor preventing increased body mass or obesity (eg, wherein the firstbacteria are Firmicutes cells).

The first configuration also provides: A method of altering the relativeratio of sub-populations of first and second bacteria in a mixedpopulation of bacteria comprising said sub-populations, wherein thefirst bacteria are host cells (eg, Bacteroidetes cells) infected by aphage and the second bacteria are not infected by said phage (or notBacteroidetes bacteria), the method comprising combining the mixedpopulation with a plurality of vectors in one or more steps forintroduction of vector nucleic acid into host cells and allowingbacterial growth in the mixed population, wherein the relative ratios ofsaid first and second bacteria is altered;

wherein each vector comprises an engineered phage-modifying (PM) CRISPRarray for introduction into a phage-infected host cell for modifying atarget nucleotide sequence of said phage in the cell,(a) wherein the PM-CRISPR array comprises one or more sequences forexpression of a PM-crRNA and a promoter for transcription of thesequence(s) in a phage-infected host cell; and(b) wherein the PM-crRNA is capable of hybridising to the phage targetsequence to guide Cas (eg, a Cas nuclease) in the infected host cell tomodify the target sequence.

In a second configuration, the invention provides:—

A host modifying (HM) CRISPR/Cas system for modifying a targetnucleotide sequence of a host cell (eg, for the use of the firstconfiguration), the system comprising components according to (i) to(iv):

-   -   (i) at least one nucleic acid sequence encoding a Cas nuclease;    -   (ii) an engineered host modifying (HM) CRISPR array comprising a        spacer sequence (HM-spacer) and repeats encoding a HM-crRNA, the        HM-crRNA comprising a sequence that is capable of hybridising to        a host target sequence to guide said Cas to the target in the        host cell to modify the target sequence;    -   (iii) an optional tracrRNA sequence or a DNA sequence for        expressing a tracrRNA sequence;    -   (iv) wherein said components of the system are split between the        host cell and at least one nucleic acid vector that can        transform the host cell, whereby the HM-crRNA guides Cas to the        target to modify the target sequence in the host cell;        wherein optionally component (i) is endogenous to the host cell.

The second configuration also provides: An engineered phage-modifying(PM) CRISPR array for use in the method of the first configuration formodifying the genome of said phage,

(a) wherein the PM-CRISPR array comprises one or more sequences forexpression of a PM-crRNA and a promoter for transcription of thesequence(s) in a phage-infected host cell; and(b) wherein the PM-crRNA is capable of hybridising to a phage genometarget sequence to guide Cas (eg, a Cas nuclease) in the infected hostcell to modify the target sequence.

In an example, the phage is a Bacteroidetes (eg, Bacteroides) phage, eg,crAssphage.

In an example, the array comprises CRISPR repeats that are functionalwith a host cell CRISPR/Cas system. This is beneficial to increaseselectivity of the array for the desired cell in a bacterial mixture.This also simplifies production of the array and vectors containing thearray of the invention as it may not be necessary to include bulkynucleotide sequences encoding one or more Cas proteins (and/or tracrRNA)required for functioning of the array in the host cell. In analternative, the array is provided with a cognate Cas9-encoding sequenceand optionally a cognate tracrRNA-encoding sequence.

In a third configuration, the invention provides:—

An engineered nucleic acid vector for modifying a bacterial host cellcomprising an endogenous CRISPR/Cas system, the vector

-   (a) comprising nucleic acid sequences for expressing a plurality of    different crRNAs (eg, single guide RNAs, ie, gRNAs) for use in a    CRISPR/Cas system or use according to the invention; and-   (b) lacking a nucleic acid sequence encoding a Cas nuclease, wherein    a first of said crRNAs is capable of hybridising to a first nucleic    acid sequence in said host cell; and a second of said crRNAs is    capable of hybridising to a second nucleic acid sequence in said    host cell, wherein said second sequence is different from said first    sequence; and-   (c) the first sequence is comprised by an antibiotic resistance gene    (or RNA thereof) and the second sequence is comprised by an    antibiotic resistance gene (or RNA thereof); optionally wherein the    genes are different;-   (d) the first sequence is comprised by an antibiotic resistance gene    (or RNA thereof) and the second sequence is comprised by an    essential or virulence gene (or RNA thereof);-   (e) the first sequence is comprised by an essential gene (or RNA    thereof) and the second sequence is comprised by an essential or    virulence gene (or RNA thereof); or-   (f) the first sequence is comprised by a virulence gene (or RNA    thereof) and the second sequence is comprised by an essential or    virulence gene (or RNA thereof).

The third configuration also provides: A nucleic acid vector (eg, aplasmid, phage or phagemid) for use in the method of the invention, thevector comprising a CRISPR array of the invention.

In a fourth configuration, the invention provides:—

A nucleic acid vector (eg, a plasmid, virus, phage or phagemid)comprising an engineered CRISPR array for modifying a target sequence ofthe genome of a host bacterial cell (eg, pathogenic bacterial cell, suchas described above) or the genome of a virus (eg, phage) in a host cell,

(a) wherein the CRISPR array comprises one or more sequences forexpression of a crRNA (eg, provided as a gRNA) and a promoter fortranscription of the sequence(s) in the host cell;(b) wherein the crRNA is capable of hybridising to the target sequenceto guide Cas (eg, a Cas nuclease) in the host cell to modify the targetsequence;(c) wherein the array is comprised by a transposon that is capable ofhorizontal transfer between first and second bacterial cells ofdifferent species.

In a fifth configuration, the invention provides:—

An engineered CRISPR nucleic acid vector comprising or consisting of amobile genetic element (MGE), wherein the MGE comprises an origin oftransfer (oriT) and a CRISPR array for modifying a target sequence ofthe genome of a host cell (eg, pathogenic bacterial cell) or the genomeof a virus (eg, prophage) in a host cell,

(a) wherein the CRISPR array comprises one or more sequences forexpression of a crRNA and a promoter for transcription of thesequence(s) in the host cell;(b) wherein the crRNA is capable of hybridising to the target sequenceto guide Cas (eg, a Cas nuclease) in the host cell to modify the targetsequence;(c) wherein the vector is capable of transfer between (i) first andsecond nucleic acid positions of a first host cell, wherein eachposition is a position on a chromosome or a plasmid and the targetsequence is comprised by the host cell, or (ii) first and second hostcells, wherein the target sequence is comprised by the first and/orsecond host cell.

In a sixth configuration, the invention provides:—

A method of controlling microbiologically influenced corrosion (MIC) orbiofouling of a substrate in an industrial or domestic system, wherein asurface of the substrate is in contact with a population of first hostcells of a first microbial species that mediates MIC or biofouling ofthe substrate, the method comprising

(i) contacting the population with a plurality of vectors that arecapable of transforming or transducing the cells, each vector comprisinga CRISPR array whereby CRISPR arrays are introduced into the host cells,wherein(a) each CRISPR array comprises one or more nucleotide sequences forexpression of a crRNA and a promoter for transcription of thesequence(s) in a host cell; and(b) each crRNA is capable of hybridising to a target sequence of a hostcell to guide Cas (eg, a Cas nuclease) in the host cell to modify thetarget sequence (eg, to cut the target sequence); the target sequencebeing a gene sequence for mediating host cell viability; and(ii) allowing expression of said cRNAs in the presence of Cas in hostcells, thereby modifying target sequences in host cells, resulting inreduction of host cell viability and control of MIC or biofouling ofsaid substrate.

In another embodiment, there is provided:—

A method of controlling microbiologically influenced corrosion (MIC) orbiofouling of a substrate comprised by a crude oil, gas orpetrochemicals recovery, processing, storage or transportationequipment, wherein a surface of the substrate is in contact with apopulation of first host cells, wherein the first host cells aresulphur- or sulphate-reducing bacteria (SRB), extracellular polymericsubstance-producing bacteria (EPSB), acid-producing bacteria (APB),sulphur- or sulphide-oxidizing bacteria (SOB), iron-oxidising bacteria(IOB), manganese-oxidising bacteria (MOB), ammonia producing bacteria(AmPB) or acetate producing bacteria (AcPB) of a first species thatmediates MIC or biofouling of the substrate, wherein the surface andcell population are in contact with a liquid selected from sea water,fresh water, a fracking liquid or liquid in a well, the methodcomprising

(i) contacting the cell population with vectors by mixing the liquidwith a plurality of vectors that are capable of transforming ortransducing first host cells, each vector comprising a CRISPR arraywhereby CRISPR arrays are introduced into the host cells, wherein

-   -   (a) each CRISPR array comprises one or more sequences for        expression of a crRNA and a promoter for transcription of the        sequence(s) in a host cell;    -   (b) each crRNA is capable of hybridising to a target sequence of        a host cell to guide Cas (eg, a Cas nuclease) in the host cell        to modify the target sequence (eg, to cut the target sequence);        the target sequence being a gene sequence for mediating host        cell viability;    -   (c) wherein each sequence of (a) comprises a sequence R1-S1-R1′        for expression and production of the respective crRNA in a first        host cell, wherein R1 is a first CRISPR repeat, R1′ is a second        CRISPR repeat, and R1 or R1′ is optional; and S1 is a first        CRISPR spacer that comprises or consists of a nucleotide        sequence that is 80% or more identical to a target sequence of a        said first host cell and        (ii) allowing expression of said cRNAs in the presence of Cas in        host cells, thereby modifying target sequences in host cells,        resulting in reduction of host cell viability and control of MIC        or biofouling of said substrate.

Other embodiments provide:—

A vector for use in the method, wherein the first cells are sulphatereducing bacteria (SRB) cells, eg, Desulfovibrio or Desulfotomaculumcells, the vector comprising one or more CRISPR arrays for targeting theSRB, wherein each array is as defined in (a)-(c). In another embodiment,there is provided: A method of controlling microbial biofouling of afluid in an industrial or domestic system, wherein the fluid comprises apopulation of first host cells of a first microbial species thatmediates said biofouling, the method comprising

(i) contacting the population with a plurality of vectors that arecapable of transforming or transducing the cells, each vector comprisinga CRISPR array whereby CRISPR arrays are introduced into the host cells,wherein(a) each CRISPR array comprises one or more nucleotide sequences forexpression of a crRNA and a promoter for transcription of thesequence(s) in a host cell; and(b) each crRNA is capable of hybridising to a target sequence of a hostcell to guide Cas (eg, a Cas nuclease) in the host cell to modify thetarget sequence (eg, to cut the target sequence); the target sequencebeing a gene sequence for mediating host cell viability; and(ii) allowing expression of said cRNAs in the presence of Cas in hostcells, thereby modifying target sequences in host cells, resulting inreduction of host cell viability and control of said biofouling. Forexample, there is provided: A method of controlling bacterial biofoulingin ballast water of a ship or boat, wherein the water comprises apopulation of first host cells of a first microbial species thatmediates said biofouling, the method comprising(i) contacting the population with a plurality of vectors that arecapable of transforming or transducing the cells, each vector comprisinga CRISPR array whereby CRISPR arrays are introduced into the host cells,wherein(a) each CRISPR array comprises one or more sequences for expression ofa crRNA and a promoter for transcription of the sequence(s) in a hostcell; and(b) each crRNA is capable of hybridising to a target sequence of a hostcell to guide Cas (eg, a Cas nuclease) in the host cell to modify thetarget sequence (eg, to cut the target sequence); the target sequencebeing a gene sequence for mediating host cell viability; and(ii) allowing expression of said cRNAs in the presence of Cas in hostcells, thereby modifying target sequences in host cells, resulting inreduction of host cell viability and control of said biofouling.

Other embodiments provide: Ballast sea water (for example, a sample ofsea water or sea water in a container) comprising CRISPR arrays, whereinthe ballast water is obtained or obtainable by the method. A ship, boat,sea container or rig comprising the ballast sea water. A vector for usein the method, wherein the first cells are Cholera (eg, vibrio, eg, O1or O139), E coli or Enterococci sp cells, the vector comprising one ormore CRISPR arrays for targeting the cells, wherein each array is asdefined in (a) and (b) of the method.

The invention also provides vectors and CRISPR arrays suitable for usein this sixth configuration or for other applications, such as formedical use, or for food or beverage treatment. To this end, there isprovided: A vector comprising a CRISPR array for introduction into abacterial host cell, wherein the bacterium is capable of water-bornetransmission, wherein

(a) the CRISPR array comprises a sequence for expression of a crRNA anda promoter for transcription of the sequence in a said host cell;(b) the crRNA is capable of hybridising to a host cell target sequenceto guide a Cas (eg, a Cas nuclease) in the host cell to modify thetarget sequence (eg, to cut the target sequence); the target sequencebeing a nucleotide sequence for mediating host cell viability;(c) wherein the sequence of (a) comprises a sequence R1-S1-R1′ forexpression and production of the crRNA, wherein R1 is a first CRISPRrepeat, R1′ is a second CRISPR repeat, and R1 or R1′ is optional; and S1is a first CRISPR spacer that comprises or consists of a nucleotidesequence that is 80% or more identical to the host cell target sequence.

Also provided are: A water or food treatment composition comprising aplurality of such vectors. A medicament for treatment or prevention of abacterial infection (eg, a Vibrio cholerae infection) in a human, themedicament comprising a plurality of such vectors. The invention alsoprovides bacterial populations, compositions, foodstuffs and beverages.For example, the foodstuff or beverage is a dairy product.

In a seventh configuration, the invention provides:—

In a first aspect:—

A method of modifying an expressible gene encoding a first Cas, themethod comprising

-   (a) combining a guide RNA (gRNA1) with the Cas gene in the presence    of first Cas that is expressed from said gene; and-   (b) allowing gRNA1 to hybridise to a sequence of said Cas gene (eg,    a promoter or a first Cas-encoding DNA sequence thereof) and to    guide first Cas to the gene, whereby the Cas modifies the Cas gene.    A first nucleic acid vector or combination of vectors, eg, for use    in the method, wherein-   (a) the first vector or a vector of said combination comprises an    expressible nucleotide sequence that encodes a guide RNA (gRNA1, eg,    a single gRNA) that is complementary to a predetermined protospacer    sequence (PS1) for guiding a first Cas to modify PS1 at a first site    (CS1), wherein PS1 is adjacent a PAM (P1) that is cognate to the    first Cas; or the expressible sequence encodes a crRNA that forms    gRNA1 with a tracrRNA; and-   (b) PS1 and P1 are sequences of an expressible first Cas-encoding    gene and PS1 is capable of being modified at CS1 by the first Cas.

These aspects of the invention are useful for regulating Cas activity,eg, in a cell or in vitro. The invention involves targeting aCas-encoding gene to restrict Cas activity, which is advantageous fortemporal regulation of Cas. The invention may also be useful in settingswhere increased stringency of Cas activity is desirable, eg, to reducethe chances for off-target Cas cutting in when modifying the genome of acell. Applications are, for example, in modifying human, animal or plantcells where off-target effects should be minimised or avoided, eg, forgene therapy or gene targeting of the cell or a tissue or an organismcomprising the cell. For example, very high stringency is required whenusing Cas modification to make desired changes in a human cell (eg, iPScell) that is to be administered to a patient for gene therapy or fortreating or preventing a disease or condition in the human. Thedisclosure provides these applications as part of the methods andproducts of the invention.

The invention also addresses the problem of restricted insert capacityin vectors, particularly in viral vectors.

Thus, an eighth configuration of the invention provides:—

A nucleic acid vector comprising more than 1.4 kb of exogenous DNAsequence encoding components of a CRISPR/Cas system, wherein thesequence comprises an engineered array or engineered sequence(optionally as described herein) for expressing one or more HM- orPM-crRNAs or gRNAs in host cells (any cell herein, eg, human, anial orbacterial or archael host cells), wherein the array or engineeredsequence does not comprise a nucleotide sequence encoding a Cas nucleasethat is cognate to the cRNA(s) or gRNA(s); optionally wherein at least2, 3 or 4 cRNAs or gRNAs are encoded by the exogenous DNA.

A nucleic acid vector comprising more than 1.4 kb or more than 4.2 kb ofexogenous DNA sequence, wherein the exogenous DNA encodes one or morecomponents of a CRISPR/Cas system and comprises an engineered array orsequence (eg, any such one described herein) for expressing one or moreHM-crRNAs or gRNAs in host cells, wherein the exogenous sequence isdevoid of a nucleotide sequence encoding a Cas nuclease that is cognateto the cRNA(s) or gRNA(s); optionally wherein at least 2 different cRNAsor gRNAs are encoded by the exogenous DNA.

A ninth configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding a Cas nuclease and host modifying        (HM) crRNAs, and    -   (b) expressing vector-encoded Cas and HM-crRNAs in host cells,        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with vector-encoded Cas in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas nuclease        to the target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population.

A tenth configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   (b) expressing HM-crRNAs in host cells,        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;        wherein the method reduces host cell population growth by at        least 5, 10-, 100, 1000, 10000, 100000 or 1000000-fold.

An eleventh configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   (b) expressing HM-crRNAs in host cells,        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;        wherein the method inhibits host cell population growth on a        surface.

A twelfth configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   (b) expressing HM-crRNAs in host cells,        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;        wherein the first species has a 16s ribosomal RNA-encoding DNA        sequence that is at least 80% identical to an 16s ribosomal        RNA-encoding DNA sequence of the host cell species, wherein the        growth of the first bacteria in the mixed population is not        inhibited by said HM-system.

A thirteenth configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   (b) expressing HM-crRNAs in host cells,        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;        wherein the mixed population of step (a) comprises a third        bacterial species.

A fourteenth configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   (b) expressing HM-crRNAs in host cells,        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;        wherein the mixed population of step (a) comprises a further        sub-population of bacterial cells of the same species as the        host cells, wherein the bacterial cells of said further        sub-population do not comprise said target sequence.

A fifteenth configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   (b) expressing HM-crRNAs in host cells,        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;        wherein each host cell comprises a plurality of said target        sequences.

A sixteenth configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   (b) expressing HM-crRNAs in host cells,        wherein Cas expression is induced in host cells, whereby said        expressed Cas and HM-crRNAs are combined in the host cells;        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population.

A seventeenth configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   (b) inducing production of HM-crRNAs in host cells,        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein expression of RNA from the engineered nucleic acid        sequence for production of HM-cRNA is inducible in the host cell        and the engineered sequence and Cas form a HM-CRISPR/Cas system,        the engineered nucleic acid sequence comprising    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population.

An eighteenth configuration of the invention provides:—

A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first bacterial sub-population and asecond bacterial sub-population wherein the first sub-populationcomprises a first microbiota species and the second sub-populationcomprises a host cell population of a second microbiota species, whereinthe second species is a different species than the first microbiotaspecies, the method comprising

-   -   (a) combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   (b) expressing HM-crRNAs in host cells,        wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and        optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;        whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population.

A nineteenth configuration of the invention provides:—

A vector that is capable of transforming a bacterial host cell, whereinthe vector is capable of accommodating the insertion of (i) a S pyogenesCas9 nucleotide sequence that is expressible in the host cell and (ii)optionally at least one HM-crRNA-encoding engineered nucleic acidsequence of the invention, for use in the method of the invention,wherein when the vector comprises (i) (and optionally (ii)) the vectoris capable of transforming the host cell and expressing a Cas (andoptionally at least one HM-crRNA (eg, a gRNA).

A twentieth configuration of the invention provides:—

A plurality of bacterial host cells, each comprising a vector of theinvention, wherein vector-encoded Cas (and optionally said HMcrRNA(s))is expressed or expressible in the host cell, wherein the bacterial cellis comprised by a mixed population of microbiota bacteria, the mixedpopulation comprising a first sub-population and a second bacterialsub-population wherein the first sub-population comprises a firstmicrobiota species and the second sub-population comprises a host cellpopulation (said plurality of bacterial host cells) of a secondmicrobiota species, wherein the second species is a different speciesthan the first microbiota species.

Herein in any configurations, for example the cRNA(s) are provided byone or more single guide RNAs (gRNAs), and in this case “CRISPR array”may refer to one or more expressible nucleotide sequences that encodesaid gRNA(s). Thus, the sequences are capable of being expressed in hostcell(s) for expressing the gRNA(s) inside the cell(s).

The invention is mainly described in terms of bacteria, but it is alsoapplicable mutatis mutandis to archaea.

Any features on one configuration herein are, in an example, combinedwith a different configuration of the invention for possible inclusionof such combination in one or more claims herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a Xylose inducible system.

FIG. 2 shows a ST1-CRISPR array.

FIG. 3 shows a spot assay on TH-agar of the strains used in this work.All strains were grown on TH-agar at 37° C. for 20 hours. Serialdilutions of overnight cultures were done in duplicate for E. coli, LLactis and S. mutans, and triplicate for both strains of S. thermophilusin order to count individual colonies.

FIGS. 4A-4C show selective growth of S. thermophilus, S. mutans, L.lactis and E. coli under different culture conditions. Tetracyclinecannot be used to selectively grown S. thermophilus LMD-9. However, 3 g1⁻¹ of PEA proved to selectively grow S. thermophilus LMD-9 whilelimiting growth of E. coli. FIG. 4A shows commencal gut bacteria. FIG.4B shows relative of target species and FIG. 4C shows a target species.

FIGS. 5A-5C illustrate construction of two xylose induction cassettes(FIGS. 5B and 5C are based on the wild type B. megaterium operon isillustrated in FIG. 5A. (Xie et al. 2013). FIG. 5B: Construction of twoxylose induction cassettes (middle, right) based on the wild type B.megaterium operon (left). (Xie et al. 2013).

FIG. 6 demonstrated characterization of the xylose inducible cassette inStreptococcus thermophilus LMD-9 with the plasmidpBAV1KT5-XylR-mCherry-Pldha. A clear response in fluorescence can beobserved with increasing amount of xylose.

FIG. 7 illustrates the design of CRISPR array inpBAV1KT5-XylR-mCherry-P_(ldha+XylA). The array contains 2 spacersequences that target S. thermophilus genes under an inducible xylosepromoter and a tracrRNA under a strong constitutive promoter P_(3A).

FIGS. 8A-8B show transformation efficiency of Streptococcus thermophilusLMD-9 with the plasmid pBAV1KT5-XylR-CRISPR-P_(ldh+XylA) (FIG. 8A) andwith pBAV1KT5-XylR-CRISPR-P_(XylA)(FIG. 8B).

FIG. 9 shows a schematic of the xylose-inducible CRISPR device. Uponinduction of xylose the CRISPR array targeting both polIII and tetA onthe S. thermophiles LMD-9 genome are expressed. Together with theconstitutively expressed tracrRNA a complex is formed with Cas9. Thiscomplex will introduce a double stranded break in the tetA and polIIIgenes in the S. thermophilus LMD-9 genome resulting in limited cellviability.

FIGS. 10A-10D show growth inhibition of Streptococcus thermophilus DSM20617(T) with the plasmid pBAV1KT5-XylR-CRISPR-PXylA (FIGS. 10A and 10C)or pBAV1KT5-XylR-CRISPR-Pldha+XylA (FIGS. 10B and 10D). Not induced(FIGS. 10A and 10B) and induced (FIGS. 10C and 10D). Picture taken after63H of incubation. Colony counts in bottom left corner (toprow: >1000, >1000, bottom row: 336, 113).

FIG. 11 shows a maximum-likelihood phylogenetic tree of 16S sequencesfrom S. thermophilus, L. lactis and E. coli.

FIGS. 12A-12F shows the selective S thermophilus growth inhibition in aco-culture of E. coli, L. lactis and S. thermophiles harboring eitherthe pBAV1KT5-XylR-CRISPR-PxylA or the pBAV1KT5-XylR-CRISPR-PldhA+XylAplasmid. No growth difference is observed between E. coli harboring thepBAV1KT5-XylR-CRISPR-PxylA or the pBAV1KT5-XylR-CRISPR-PldhA+XylAplasmid (FIGS. 12B and 12E). However, S. thermophiles (selectively grownon TH agar supplemented with 2.5 gl-1 PEA, FIGS. 12C and 12F) shows adecrease in transformation efficiency between thepBAV1KT5-XylR-CRISPR-PxylA (strong) or thepBAV1KT5-XylR-CRISPR-PldhA+XylA (weak) plasmid as we expected. We thusdemonstrated a selective growth inhibition of the target S. thermophilussub-population in the mixed population of cells. Colony counts in bottomleft corner (top row: >1000, >1000, 68, bottom row: >1000, >1000, 32).

FIG. 13 shows regulators controlling the expression of spCas9 and theself-targeting sgRNA targeting the ribosomal RNA subunit 16s.

FIG. 14 shows specific targeting of E. coli strain by an exogenousCRISPR-Cas system. The sgRNA target the genome of K-12 derived E. colistrains, like E. coli TOP10, while the other strain tested wasunaffected.

FIG. 15 shows spot assay with serial dilutions of individual bacterialspecies used in this study and mixed culture in TH agar withoutinduction of CRISPR-Cas9 system.

FIG. 16 shows spot assay of the dilution 10³ on different selectivemedia. TH with 2.5 g 1⁻¹ PEA is a selective media for B. subtilis alone.MacConkey supplemented with maltose is a selective and differentialculture medium for bacteria designed to selectively isolateGram-negative and enteric bacilli and differentiate them based onmaltose fermentation. Therefore TOP10 ΔmalK mutant makes white colonieson the plates while Nissle makes pink colonies; A is E coli ΔmalK, B isE coli Nissile, C is B subtilis, D is L lactis, E is mixed culture; theimages at MacConkey−/B and E appear pink; the images at MacConkey+/B andE appear pink. FIG. 17 shows selective growth of the bacteria used inthis study on different media and selective plates.

DETAILED DESCRIPTION Inhibiting Microbial Population Growth & AlteringMicrobial Ratios

The invention relates to methods, uses, systems, arrays, cRNAs, gRNAsand vectors for inhibiting bacterial population growth or altering therelative ratio of sub-populations of first and second bacteria in amixed population of bacteria, eg, for altering human or animalmicrobiomes, such as for the alteration of the proportion ofBacteroidetes (eg, Bacteroides), Firmicutes and/or gram positive ornegative bacteria in microbiota of a human. See, for example, the firstto third configurations described herein. The invention, for example,involves modifying one or more target nucleotide sequences of a hostbacterial cell, eg, a Bacteroidetes cell or Firmicutes cell.

There have been a number of studies pointing out that the respectivelevels of the two main intestinal phyla, the Bacteroidetes and theFirmicutes, are linked to obesity, both in humans and in germfree mice.The authors of the studies deduce that carbohydrate metabolism is theimportant factor. They observe that the microbiota of obese individualsare more heavily enriched with bacteria of the phylum Firmicutes andless with Bacteroidetes, and they surmise that this bacterial mix may bemore efficient at extracting energy from a given diet than themicrobiota of lean individuals (which have the opposite proportions). Insome studies, they found that the relative abundance of Bacteroidetesincreases as obese individuals lose weight and, further, that when themicrobiota of obese mice are transferred to germfree mice, these micegain more fat than a control group that received microbiota from leanmice. See, eg, Turnbaugh, P. J., R. E. Ley, M. A. Mahowald, V. Magrini,E. R. Mardis, and J. I. Gordon. 2006, “An obesity-associated gutmicrobiome with increased capacity for energy harvest”, Nature444:1027-1131.

Concepts

The invention provides the following concepts involving a host celltarget:—1. Use of a host modifying (HM) CRISPR/Cas system for killing orreducing the growth of bacterial host cells, for each host cell thesystem comprising components according to (i) to (iv):—

(i) at least one nucleic acid sequence encoding a Cas nuclease;(ii) an engineered host modifying (HM) CRISPR array comprising a spacersequence (HM-spacer) and repeats encoding a HM-crRNA, the HM-crRNAcomprising a sequence that hybridises to a host cell target sequence toguide said Cas to the target in the host cell to modify the targetsequence;(iii) an optional tracrRNA sequence or a DNA sequence expressing atracrRNA sequence;(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that transforms the host cell,whereby the HM-crRNA guides Cas to the target to modify the targetsequence in the host cell;wherein the Cas nuclease is endogenous to the host cell; and wherein thetarget sequence is modified by the Cas whereby the host cell is killedor host cell growth is reduced.

Concept 1 alternatively provides:

Use of a host modifying (HM) CRISPR/Cas system for altering the relativeratio of sub-populations of first and second bacteria in a mixedpopulation of bacteria, the second bacteria comprising host cells, foreach host cell the system comprising components according to (i) to(iv):—

(i) at least one nucleic acid sequence encoding a Cas nuclease;(ii) an engineered host modifying (HM) CRISPR array comprising a spacersequence (HM-spacer) and repeats encoding a HM-crRNA, the HM-crRNAcomprising a sequence that hybridises to a host cell target sequence toguide said Cas to the target in the host cell to modify the targetsequence;(iii) an optional tracrRNA sequence or a DNA sequence expressing atracrRNA sequence;(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that transforms the host cell,whereby the HM-crRNA guides Cas to the target to modify the targetsequence in the host cell; wherein optionally the Cas nuclease isendogenous to the host cell; and wherein the target sequence is modifiedby the Cas whereby the host cell is killed or host cell growth isreduced.

Concept 1 also provides: A method of altering the relative ratio ofsub-populations of first and second bacteria in a mixed population ofbacteria, the second bacteria comprising host cells, and the methodcomprising combining the mixed population with of a host modifying (HM)CRISPR/Cas system whereby second bacteria host cells are killed or thegrowth of said cells is reduced thereby altering said ratio, wherein foreach host cell the system comprises components according to (i) to(iv):—

(i) at least one nucleic acid sequence encoding a Cas nuclease;(ii) an engineered host modifying (HM) CRISPR array comprising a spacersequence (HM-spacer) and repeats encoding a HM-crRNA, the HM-crRNAcomprising a sequence that hybridises to a host cell target sequence toguide said Cas to the target in the host cell to modify the targetsequence;(iii) an optional tracrRNA sequence or a DNA sequence expressing atracrRNA sequence;(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that transforms the host cell,whereby the HM-crRNA guides Cas to the target to modify the targetsequence in the host cell;wherein optionally the Cas nuclease is endogenous to the host cell; andwherein the target sequence is modified by the Cas whereby the host cellis killed or host cell growth is reduced.Concept 1 also provides:—

Use of a host modifying (HM) CRISPR/Cas system for altering the relativeratio of sub-populations of first and second bacteria in a mixedpopulation of bacteria, the second bacteria comprising a plurality ofhost cells each comprising a target protospacer sequence, for each hostcell the system comprising components (ii) and (iii) defined above, thesystem further comprising at least one nucleic acid sequence encoding aCas nuclease; wherein said component (ii) and said Cas-encoding sequenceare comprised by at least one nucleic acid vector that transforms thehost cell, whereby the HM-crRNA encoded by (i) guides Cas to the targetto modify the target sequence in the host cell; wherein the Cas nucleaseis endogenous to the host cell; and wherein the target sequence ismodified by the Cas whereby the host cell is killed or host cell growthis reduced.

In an embodiment, the growth of first bacteria is not inhibited; or thegrowth inhibition of said host cells is at least 2×, 3×, 4×, 5×, 6×, 7×,8×, 9×, 10×, 50×, 100× or 1000× the growth inhibition of the firstcells. The growth inhibition can be calculated as a fold-inhibition oras a percentage inhibition (as described herein). In another example,inhibition is measured in a culture sample by a spectrophotometer,wherein light absorbance (eg, at OD₆₀₀) is determined at the start andend of a predetermined crRNA/gRNA treatment period (see the descriptionof such a period herein when determining inhibition by fold orpercentage). In an example, the increase in absorbance (comparing theabsorbance at the beginning of the predetermined period with absorbanceat the end of that period) for the host cell sample is less than for thecontrol sample (which has not been exposed to said cRNA or gRNA), eg,the increase for the former is at least 10, 100, 1000, 10000 or 100000times lower than for the latter (eg, determined as OD₆₀₀). In anexample, the determination of growth inhibition (ie, the end of thepredetermined period) is made at the mid-exponential growth phase ofeach sample (eg, 6-7 hours after the start of the predetermined period).

In an example, the host cells are comprised by a microbiota populationcomprised by an organism or environment (eg, a waterway microbiota,water microbiota, human or animal gut microbiota, human or animal oralcavity microbiota, human or animal vaginal microbiota, human or animalskin or hair microbiota or human or animal armpit microbiota), thepopulation comprising first bacteria that are symbiotic or commensalwith the organism or environment and second bacteria comprising saidhost cells, wherein the host cells are detrimental (eg, pathogenic) tothe organism or environment. In an embodiment, the population is exvivo.

The ratio of the first bacteria sub-population to the second bacteriasub-population is increased.

Concept 1 also provides a use for inhibiting host cell growth asdescribed further below.

2. A host modifying (HM) CRISPR/Cas system for modifying a targetnucleotide sequence of a host cell (eg, for the use of concept 1), thesystem comprising components according to (i) to (iv):—

(i) at least one nucleic acid sequence encoding a Cas nuclease;(ii) an engineered host modifying (HM) CRISPR array comprising a spacersequence (HM-spacer) and repeats encoding a HM-crRNA, the HM-crRNAcomprising a sequence that is capable of hybridising to a host targetsequence to guide said Cas to the target in the host cell to modify thetarget sequence;(iii) an optional tracrRNA sequence or a DNA sequence for expressing atracrRNA sequence;(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that can transform the hostcell, whereby the HM-crRNA guides Cas to the target to modify the targetsequence in the host cell;wherein optionally component (i) is endogenous to the host cell.

In an alternative, HM-crRNA and tracrRNA are comprised by a single guideRNA (gRNA).

By harnessing endogenous Cas nuclease, embodiments of the invention useendogenous Cas nuclease activity (ie, without the need for prior geneticmodification of the host cell to activate or enhance the nucleaseactivity). Thus, in an example, the Cas nuclease is encoded by awild-type gene of the host cell. In an example, the nuclease is activeto achieve the cell killing or growth reduction without inhibition of anendogenous Cas nuclease (or Cas nuclease gene) repressor in the hostcell. Thus, the invention can address wild-type bacterial populationswithout the need for prior manipulation to make bring about effectiveCas-mediated cell killing or growth reduction. Thus, the population canbe exposed to the cRNA when the population is in its wild-typeenvironment (such as a waterway or comprised by a human or animalmicrobiome).

In an example, the second bacteria are Bacteroidetes (eg, Bacteroides)cells. In an example, the second bacteria are Firmicutes cells. The use,system or method is, for example, used to alter the ratios in a gutmicrobiota population (eg, ex vivo or in vivo), which is for example fortreating or preventing increased body mass or obesity (eg, wherein thesecond bacteria are Firmicutes cells).

In an example, the use, method, system, vector, engineered nucleotidesequence, cRNA or gRNA is for therapeutically or prophylacticallyrebalancing microbiota of a human or non-human animal comprising themixed population, eg for treating or preventing obesity, diabetes IBD, aGI tract condition or an oral cavity condition.

In an example, the microbiota mentioned herein is microbiota of a humanor animal microbiome (eg, gut, vaginal, scalp, armpit, skin bloodstream,throat or oral cavity microbiome).

In an example, the microbiota mentioned herein is an armpit microbiotaand the use, method, system, vector, engineered nucleotide sequence,cRNA or gRNA is for preventing or reducing body odour of a human.

In an example, the host cell population or mixed population is harbouredby a beverage or water (eg, a waterway or drinking water) for humanconsumption.

In an example, the use, method, system, vector, engineered nucleotidesequence, cRNA or gRNA is for reducing pathogenic infections or forre-balancing gut or oral microbiota eg, for treating or preventingobesity or disease in a human or animal. For example, the use, method,system, vector, engineered nucleotide sequence, cRNA or gRNA is forknocking-down Clostridium dificile bacteria in a gut microbiota.

In an example, the first bacteria are Bacteroides bacteria and thesecond bacteria are Firmicutes or pathogenic bacteria, eg, gut bacteria.In an example, the host cells or second bacteria are Firmicutes cells,eg, selected from Streptococcus (eg, thermophilus and/or pyogenes),Bacillus, Lactobacillus, Listeria, Clostridium, Heliobacterium andStaphylococcus cells. In an example, the mixed population containsBacteroides and metronidazole (MTZ)-resistant C dificile strain 630sub-populations, wherein the host cells comprise said C dificile cells.

In an example, the host cell population, mixed population or system iscomprised by a composition (eg, a beverage, mouthwash or foodstuff) foradministration to a human or non-human animal for populating andrebalancing the gut or oral microbiota thereof.

In an example, the product of the use or method, or the system, vector,engineered nucleotide sequence, cRNA or gRNA is for administration to ahuman or non-human animal by mucosal, gut, oral, intranasal,intrarectal, intravaginal, ocular or buccal administration.

In an example of any configuration herein, the mixed population (priorto combining with the array, gRNA, crRNA or engineered sequence) is asample of a microiota of a human or animal subject, eg, a gut or anyother microbiota disclosed herein or a microbiota of any microbiomedisclosed herein. In an example, in this instance the product of the useof the invention is a modified microbiota population that is useful foran treatment or therapy of a human or animal subject, as disclosedherein.

3. The system of concept 2, wherein the vector or vectors lack a Cas(eg, a Cas9) nuclease-encoding sequence.

4. The use, method or system of any preceding concept, wherein each hostcell is of a strain or species found in human microbiota, optionallywherein the host cells are mixed with cells of a different strain orspecies, wherein the different cells are Enterobacteriaceae or bacteriathat are probiotic, commensal or symbiotic with humans (eg, in the humangut. In an example, the host cell is a Firmicutes, eg, Streptococcus,cell.

5. The use, method or system of any preceding concept for the alterationof the proportion of Bacteroidetes (eg, Bacteroides) bacteria in a mixedbacterial population (eg, in a human, such as in human microbiota).

6. The use, method or system of concept 5 for increasing the relativeratio of Bacteroidetes versus Firmicutes.

7. The use, method or system of any preceding concept, wherein said Casnuclease is provided by an endogenous Type II CRISPR/Cas system of thecell.

8. The use, method or system of any preceding concept, wherein component(iii) is endogenous to the host cell.

9. The use, method or system of any preceding concept, wherein thetarget sequence is comprised by an antibiotic resistance gene, virulencegene or essential gene of the host cell.

10. The use, method or system of any preceding concept, the array beingcomprised by an antibiotic composition, wherein the array is incombination with an antibiotic agent.

11. The use, method or system of any preceding concept, whereinalternatively HM-crRNA and tracrRNA are comprised by a single guide RNA(gRNA), eg provided by the vector.

12. The use, method or system of any preceding concept, wherein the hostcell comprises a deoxyribonucleic acid strand with a free end (HM-DNA)encoding a HM-sequence of interest and/or wherein the system comprises asequence encoding the HM-DNA, wherein the HM-DNA comprises a sequence orsequences that are homologous respectively to a sequence or sequences inor flanking the target sequence for inserting the HM-DNA into the hostgenome (eg, into a chromosomal or episomal site).

13. An engineered nucleic acid vector for modifying a bacterial hostcell comprising an endogenous CRISPR/Cas system, the vector

(a) comprising nucleic acid sequences for expressing a plurality ofdifferent crRNAs (eg, gRNAs) for use in a CRISPR/Cas system, method oruse according to any preceding concept; and(b) optionally lacking a nucleic acid sequence encoding a Cas nuclease,wherein a first of said crRNAs is capable of hybridising to a firstnucleic acid sequence in said host cell; and a second of said crRNAs iscapable of hybridising to a second nucleic acid sequence in said hostcell, wherein said second sequence is different from said firstsequence; and(c) the first sequence is comprised by an antibiotic resistance gene (orRNA thereof) and the second sequence is comprised by an antibioticresistance gene (or RNA thereof); optionally wherein the genes aredifferent;(d) the first sequence is comprised by an antibiotic resistance gene (orRNA thereof) and the second sequence is comprised by an essential orvirulence gene (or RNA thereof);(e) the first sequence is comprised by an essential gene (or RNAthereof) and the second sequence is comprised by an essential orvirulence gene (or RNA thereof); or(f) the first sequence is comprised by a virulence gene (or RNA thereof)and the second sequence is comprised by an essential or virulence gene(or RNA thereof).

14. The vector of concept 13 inside a host cell comprising one or moreCas that are operable with cRNA (eg, single guide RNA) encoded by thevector.

15. The use, method, system or vector of any preceding concept, whereinthe HM-CRISPR array comprises multiple copies of the same spacer.

16. The use, method, system or vector of any preceding concept, whereinthe vector(s) comprises a plurality of HM-CRISPR arrays.

17. The use, method, system or vector of any preceding concept, whereineach vector is a plasmid, cosmid, virus, a virion, phage, phagemid orprophage.

18. The use, method, system or vector of any preceding concept, whereinthe system or vector comprises two, three or more of copies of nucleicacid sequences encoding crRNAs (eg, gRNAs), wherein the copies comprisethe same spacer sequence for targeting a host cell sequence (eg, avirulence, resistance or essential gene sequence).

19. The use, method, system or vector of concept 18, wherein the copiesare split between two or more vector CRISPR arrays.

20. A bacterial host cell comprising a system or vector recited in anypreceding concept.

21. The system, vector or cell of any one of concepts 2 to 20 incombination with an antibiotic agent (eg, a beta-lactam antibiotic).

22. The use, method, system, vector or cell of any preceding concept,wherein the or each host cell is a Staphylococcus, Streptococcus,Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio or Clostridiumhost cell. In an example, the or each host cell is a Firmicutes cell,eg, a Staphylococcus, Streptococcus, Listeria or Clostridium cell.

In an example, each CRISPR array comprises a sequence R1-S1-R1′ forexpression and production of the respective crRNA (eg, comprised by asingle guide RNA) in the host cell, (i) wherein R1 is a first CRISPRrepeat, R1′ is a second CRISPR repeat, and R1 or R1′ is optional; and(ii) S1 is a first CRISPR spacer that comprises or consists of anucleotide sequence that is 95% or more identical to said targetsequence.

In an example, R1 and R1′ are at least 95% identical respectively to thefirst and second repeat sequences of a CRISPR array of the second hostcell species. In an example, R1 and R1′ are at least 95% (eg, 96, 97,98, 99 or 100%) identical respectively to the first (5′-most) and second(the repeat immediately 3′ of the first repeat) repeat sequences of aCRISPR array of said species, eg, of a said host cell of said species.In an example, R1 and R1′ are functional with a Type II Cas9 nuclease(eg, a S thermophilus, S pyogenes or S aureus Cas9) to modify the targetin a said host cell.

An alternative Concept 1 use of invention provides the following, asdemonstrated by the worked experimental Example:

The use of wild-type endogenous Cas nuclease activity of a bacterialhost cell population to inhibit growth of the population, wherein eachhost cell has an endogenous CRISPR/Cas system having wild-type Casnuclease activity, the use comprising transforming host cells of thepopulation, wherein each transformed host cell is transformed with anengineered nucleotide sequence for providing host modifying (HM) cRNA orguide RNA (gRNA) in the host cell, the HM-cRNA or gRNA comprising asequence that is capable of hybridising to a host cell targetprotospacer sequence for guiding endogenous Cas to the target, whereinthe cRNA or gRNA is cognate to an endogenous Cas nuclease of the hostcell that has said wild-type nuclease activity and followingtransformation of the host cells growth of the population is inhibited.

In the worked Example below, inhibition was addressed in a bacterialpopulation (a gram positive Firmicutes) on a solid surface. A >10-foldinhibition of host cell population growth was achieved. Targeting wasdirected to an antibiotic resistance gene and an essential gene. Theinvention will be useful in inhibiting the growth ofantibiotic-resistant bacteria, wherein the target sequence is a sequenceof an antibiotic resistance gene. In an example, co-administration ofthe engineered nucleotide sequence with the antibiotic may be effective.This may provide more complete treatment or prevention of host cellinfection in human or animal subjects and/or enable the reduction oftherapeutically-effective antibiotic dose for administration to a humanor animal. This is useful in view of the increasing worry regardingover-administration of antibiotics and the development of resistance inhuman and animal populations.

The demonstration of the invention's ability to inhibit host cell growthon a surface is important and desirable in embodiments where theinvention is for treating or preventing diseases or conditions mediatedor caused by microbiota as disclosed herein in a human or animalsubject. Such microbiota are typically in contact with tissue of thesubject (eg, gut, oral cavity, lung, armpit, ocular, vaginal, anal, ear,nose or throat tissue) and thus we believe that the demonstration ofactivity to inhibit growth of a microbiota bacterial species(exemplified by Streptococcus) on a surface supports this utility.

In an example, wild-type host cell endogenous Cas9 or cfp1 activity isused. The engineered nucleotide sequence may not be in combination withan exogenous Cas nuclease-encoding sequence.

In an example, the host cells are wild-type (eg, non-engineered)bacterial cells. In another example, the host cells are engineered (suchas to introduce an exogenous nucleotide sequence chromosomally or tomodify an endogenous nucleotide sequence, eg, on a chromosome or plasmidof the host cell), and wherein the host cells comprise an endogenousCRISPR/Cas system having wild-type Cas nuclease activity that isoperable with the crRNA or gRNA. In an example, the formation ofbacterial colonies of said host cells is inhibited following saidtransformation. In an example, proliferation of host cells is inhibitedfollowing said transformation. In an example, host cells are killedfollowing said transformation.

By “cognate to” it is intended that the endogenous Cas is operable withcrRNA or gRNA sequence to be guided to the target in the host cell. Theskilled addressee will understand that such Cas guiding is generally afeature of CRISPR/Cas activity in bacterial cells, eg, wild-typeCRISPR/Cas activity in bacterial cells having endogenous activewild-type CRISPR/Cas systems.

By “wild-type” Cas activity it is intended, as will be clear to theskilled addressee, that the endogenous Cas is not an engineered Cas orthe cell has not been engineered to de-repress the endogenous Casactivity. This is in contrast to certain bacteria where Cas nucleaseactivity is naturally repressed (ie, there is no wild-type Cas nucleaseactivity or none that is useful for the present invention, which on thecontrary is applicable to addressing wild-type host cells in situ forexample where the endogenous Cas activity can be harnessed to effectcell population growth inhibition).

In an example, inhibition of host cell population growth is at least 2,3, 4, 5, 6, 7, 8, 9 or 10-fold compared to the growth of said host cellsnot exposed to said engineered nucleotide sequence. For example, growthinhibition is indicated by a lower bacterial colony number of a firstsample of host cells (alone or in a mixed bacterial population) by atleast 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold compared to the colony number ofa second sample of the host cells (alone or in a mixed bacterialpopulation), wherein the first cells have been transformed by saidengineered nucleotide sequence but the second sample has not beenexposed to said engineered nucleotide sequence. In an embodiment, thecolony count is determined 12, 24, 36 or 48 hours after the first samplehas been exposed to the engineered sequence. In an embodiment, thecolonies are grown on solid agar in vitro (eg, in a petri dish). It willbe understood, therefore, that growth inhibition can be indicated by areduction (<100% growth compared to no treatment, ie, control samplegrowth) in growth of cells or populations comprising the targetsequence, or can be a complete elimination of such growth. In anexample, growth of the host cell population is reduced by at least 10,20, 30, 40, 50, 60, 70, 80, 90 or 95%, ie, over a predetermined timeperiod (eg, 24 hours or 48 hours following combination with the cRNA orgRNA in the host cells), ie, growth of the host cell population is atleast such percent lower than growth of a control host cell populationthat has not been exposed to said cRNA or gRNA but otherwise has beenkept in the same conditions for the duration of said predeterminedperiod. In an example, percent reduction of growth is determined bycomparing colony number in a sample of each population at the end ofsaid period (eg, at a time of mid-exponential growth phase of thecontrol sample). For example, after exposing the test population to thecrRNA or gRNA a time zero, a sample of the test and control populationsis taken and each sample is plated on an agar plate and incubated underidentical conditions for said predetermined period. At the end of theperiod, the colony number of each sample is counted and the percentagedifference (ie, test colony number divided by control colony number andthen times by 100, and then the result is subtracted from 100 to givepercentage growth reduction). The fold difference is calculated bydividing the control colony number by the test colony number.

Inhibition of population growth can be indicated, therefore, by areduction in proliferation of host cell number in the population. Thismay be due to cell killing by the nuclease and/or by downregulation ofhost cell proliferation (division and/or cell growth) by the action ofthe nuclease on the target protospacer sequence. In an embodiment of atreatment or prevention as disclosed herein, host cell burden of thehuman or animal subject is reduced, whereby the disease or condition istreated (eg, reduced or eliminated) or prevented (ie, the risk of thesubject developing the disease or condition) is reduced or eliminated.

The invention is useful for targeting wild-type bacterial populationsfound naturally in the environment (eg, in water or waterways, coolingor heating equipment), comprised by beverages and foodstuffs (orequipment for manufacturing, processing or storing these) or wild-typebacterial populations comprised by human or animal microbiota. Thus, theinvention finds utility in situations when pre-modification of hostcells to make them receptive to killing or growth inhibition is notpossible or desirable (eg, when treatment in situ of microbiota in thegut or other locations of a subject is desired). In another application,the invention finds utility for producing ex vivo a medicament foradministration to a human or animal subject for treating or preventing adisease or condition caused or mediated by the host cells, wherein themedicament comprises a modified mixed bacterial population (eg, obtainedfrom faeces or gut microbiota of one or more human donors) which is theproduct of the use or method of the invention, wherein the populationcomprises a sub-population of bacteria of a species or strain that isdifferent to the species or strain of the host cells. The formersub-population cells do not comprise the target and thus are notmodified by the use or method. Thus, for example, the method can be usedto reduce the proportion of a specific Firmicutes sub-population andspare Bacteroidetes in the mixed population, eg, for producing amedicament for treating or preventing a metabolic or GI condition ordisease disclosed herein. In this way, the invention can provide amodified bacterial transplant (eg, a modified faecal transplant)medicament for such use or for said treatment or prevention in a humanor animal. For example, the method can be used to modify one or moremicrobiota in vitro to produce a modified collection of bacteria foradministration to a human or animal for medical use (eg, treatment orprevention of a metabolic condition (such as obesity or diabetes) or aGI tract condition (eg, any such condition mentioned herein) or a cancer(eg, a GI tract cancer)) or for cosmetic or personal hygiene use (eg,for topical use on a human, eg, for reducing armpit or other body odourby topical application to an armpit of a human or other relevantlocation of a human). In another example, the array, crRNA, gRNA orengineered nucleotide sequence is administered to a human or animal andthe host cells are harboured by the human or animal, eg, comprised by amicrobiota of the human or animal (such as a gut microbiota or any othertype of micriobiota disclosed herein). In this way, a disease orcondition mediated or caused by the host cells can be treated orprevented. In an example, the transformation is carried out in vitro andoptionally the array, crRNA, gRNA or engineered nucleotide sequence iscomprised by nucleic acid that is electroporated into host cells. In anexample, the nucleic acid are RNA (eg, copies of the gRNA). In anotherexample, the nucleic acid are DNA encoding the crRNA or gRNA forexpression thereof in host cells.

Thus, in an example, the invention provides an engineered nucleotidesequence for providing host cell modifying (HM) cRNA or guide RNA (gRNA)in a population of wild-type bacterial host cells comprised by amicrobiota of a human or animal subject for treating or preventing adisease or condition mediated or caused by host cells of the microbiotaof the subject, the cRNA or gRNA comprising a sequence that is capableof hybridising to a host cell target protospacer sequence for guidingCas to the target, wherein the cRNA or gRNA is cognate to an endogenoushost cell Cas nuclease that has wild-type nuclease activity, whereinfollowing transformation of host cells growth of the population isinhibited and the disease or condition is treated or prevented.

In an example, the engineered nucleotide sequence comprises a HM-CRISPRarray as defined herein. In an example, the engineered nucleotidesequence encodes a single guide RNA. In an example, the engineerednucleotide sequence is a guide RNA (eg, a singe guide RNA) or crRNA. Inan example, the engineered sequence is comprised by a bacteriophage thatis capable of infecting the host cells, wherein the transformationcomprises transduction of the host cells by the bacteriophage. Thebacteriophage can be a bacteriophage as described herein. In an example,the engineered nucleotide sequence is comprised by a plasmid (eg, aconjugative plasmid) that is capable of transforming host cells. Theplasmid can be a plasmid as described herein. In an example, theengineered nucleotide sequence is comprised by a transposon that iscapable of transfer into and/or between host cells. The transposon canbe a transposon as described herein.

Any use or method of the invention can comprise transforming host cellswith nucleic acid vectors for producing cRNA or gRNA in the cells. Forexample, the vectors or nucleic acid comprising the engineerednucleotide sequence are administered orally, intravenously, topically,ocularly, intranasally, by inhalation, by rectal administration, in theear, by vaginal administration or by any other route of administrationdisclosed herein or otherwise to a human or animal comprising the mixedbacterial population (eg, as part of microbiota of the human or animal),wherein the administration transforms the host cells with the vectors ornucleic acid.

In an example, the host cell population is ex vivo. In an example, themixed population is comprised by a human or animal subject and a hostcell infection in the subject is treated or prevented.

In an example, the first and second bacteria are comprised by amicrobial consortium wherein the bacteria live symbiotically. In anexample, the consortium is a human or animal microbiota; in an examplethe consortium is comprised by a human or animal (eg, wherein the use,system, engineered sequence, vector or cell is for treating infection byhost cells of the consortium in the human or animal, eg, wherein thehost cells mediate or cause antibiotic resistance or a deleteriousdisease or condition in the human or animal). The species (E coli, Llactis and S thermophilus) used in the worked Example below are strainsthat co-exist symbiotically in human and animal gut microbiota. TheExample also addresses targeting in a mixed gram positive and gramnegative bacterial population. Additionally, the Example addresses apopulation of Firmicutes (S thermophilus) and a population ofEnterobacteriaceae (E coli), both of which are found in humanmicrobiota. Other examples of Enterobacteriaceae are Salmonella,Yersinia pestis, Klebsiella, Shigella, Proteus, Enterobacter, Serratia,and Citrobacter.

In an example, the method, use, engineered nucleotide sequence, array,crRNA, gRNA, vector or system is for treating host cell infection in ahuman gut microbiota population, optionally the population alsocomprising first bacteria that are human commensal gut bacteria and/orEnterobacteriaceae, eg, wherein the host cells and commensal cells(first and second bacteria) live symbiotically in human gut microbiota.

In an example the use or system is for the alteration of the proportionof Bacteroidetes bacteria in a mixed bacterial population comprisingBacteroidetes bacteria and other bacteria. For example, for increasingthe relative ratio of Bacteroidetes versus one, more or all Firmicutes(eg, versus Streptococcus) in the population. In this case, the hostcells can be Firmicutes cells comprising the target(s). In an example,the population is a bacterial population of a microbiota comprised by ahuman or animal subject and the method, use, engineered nucleotidesequence, vector or system is for (i) treating an infection in thesubject by said host cells comprised (eg, comprised by the mixedpopulation); (ii) treating or preventing in the subject a condition ordisease mediated by said host cells; (iii) reducing body odour of thehuman that is caused or mediated by said host cells; or (iv) personalhygiene treatment of the human. In an example, the engineered nucleotidesequence, array, crRNA, gRNA or vector of the invention is for use insuch a system or use of the invention.

In an example, the condition or disease is a metabolic orgastrointestinal disease or condition, eg, obesity, IBD, IBS, Crohn'sdisease or ulcerative colitis. In an example, the condition or diseaseis a cancer, eg, a solid tumour or a GI cancer (eg, stomach cancer),liver cancer or pancreatic cancer. In an example, the condition isresistance or reduced responsiveness to an antibiotic (eg, anyantibiotic disclosed herein).

In an example, the cell comprises an endogenous RNase III that isoperable with component (ii) in the production of said HM-crRNA in thecell. In an alternative, one or more of the vectors comprises anucleotide sequence encoding such a RNase III for expression of theRNase III in the host cell.

In an example, the essential gene (comprising the target) encodes a DNApolymerase of the cell. This is exemplified below. In an example of theuse, system, vector or cell, array, cRNA or gRNA comprises a sequencethat is capable of hybridising to a host cell target protospacersequence that is a adjacent a NGG, NAG, NGA, NGC, NGGNG, NNGRRT orNNAGAAW protospacer adjacent motif (PAM), eg, a AAAGAAA or TAAGAAA PAM(these sequences are written 5′ to 3′). In an embodiment, the PAM isimmediately adjacent the 3′ end of the protospacer sequence. In anexample, the Cas is a S aureus, S theromophilus or S pyogenes Cas. In anexample, the Cas is Cpf1 and/or the PAM is TTN or CTA.

In an example the engineered nucleotide sequence, crRNA, gRNA or arrayis in combination with an antibiotic agent, eg, wherein the target iscomprised by an antibiotic resistance gene wherein the antibiotic issaid agent. In embodiment, the host cells are sensitive to theantibiotic. For example, there may be insufficient sensitivity to usethe antibiotic to eradicate infection of presence of the host cells (eg,in a human or manufacturing vessel/equipment comprising the population),but the antibiotic can dampen down or reduce host cell sub-populationsize or growth whilst further killing or growth inhibition is effectedusing Cas modification (eg, target cutting) according to the invention.

The invention provides the use, system, array, crRNA, gRNA, engineerednucleotide sequence, vector or cell for a method of antibiotic (firstantibiotic) treatment of an infection of said host cells in a human oranimal subject, wherein an antibiotic resistance gene (for resistance tothe first antibiotic) is Cas-targeted by the system or vector in hostcells, wherein the method comprises administering the system, array,crRNA, gRNA, engineered nucleotide sequence, vector or cell and theantibiotic to the subject. The gene is downregulated, ie, expression ofa protein product encoded by the gene is reduced or eliminated in thehost cell, whereby antibiotic resistance is downregulated. The infectionis reduced or prevented in the subject. In an example, the antibiotic isadministered simultaneously with the system, array, crRNA, gRNA,engineered nucleotide sequence, vector or cell; in another example, theadministration is sequential (eg, the antibiotic before the system,array, crRNA, gRNA, engineered nucleotide sequence, vector or cell).This feature of the invention can be useful for enhancing antibiotictreatment in the subject, eg, when antibiotic alone is not fullyeffective for treating such a host cell infection. The antibiotic can beany antibiotic disclosed herein, eg, tetracycline.

In an example, each engineered nucleotide sequence or vector comprises asaid CRISPR array or a sequence encoding a said crRNA or gRNA andfurther comprises an antibiotic resistance gene (eg, kanamycinresistance), wherein the HM-crRNA or gRNA does not target the antibioticresistance gene. In an example, the target sequence is comprised by anantibiotic resistance gene of the host cell, wherein the antibiotic isdifferent from the first antibiotic (eg, kanamycin). In this way, thesystem, engineered sequence or vector is able to target the host withouttargeting itself. By exposing the host cells to the first antibiotic,one can promote retention of the engineered sequence or vector thereinby positive selection pressure since cells containing the firstantibiotic resistance gene will have a survival advantage in thepresence of the first antibiotic (when host cells that are nottransformed by the engineered sequence or vectors are not resistant tothe first antibiotic). Thus, an example provides: The use of theinvention comprising exposing the host cell or mixed population to saidantibiotic (eg, kanamycin) and said engineered sequence or vector(s),for promoting maintenance of cRNA or gRNA-encoding sequences in hostcells; or the system, engineered sequence, array or vector of theinvention is in combination with said antibiotic.

In an example the sequence encoding the cRNA or gRNA or the component(ii) is under a constitutive promoter (eg, a strong promoter) operablein the host cell species, or an inducible promoter. In an examplecomponent (iii) is under a constitutive promoter operable in the hostcell species, or an inducible promoter.

In an example, the or each host cell is a gram positive cell. In anotherexample, the or each host cell is a gram positive cell.

In an example the method, use, system, engineered sequence or vector isfor treating host cell infection in a human gut microbiota population,optionally the population comprising human commensal gut bacteria (ie,gut bacteria that are commensal with humans).

In an example of the method, use, system, array, crRNA, gRNA, engineeredsequence or vector, the host cells are comprised by a mixed bacterialpopulation comprised by a human or animal subject and the method, use,system, array, crRNA, gRNA, engineered sequence or vector is for (i)treating an infection in the subject by said host cells comprised by themixed population; (ii) treating or preventing in the subject a conditionor disease mediated by said host cells; (iii) reducing body odour of thehuman that is caused or mediated by said host cells; or (iv) personalhygiene treatment of the human.

In an example of the method, use, system, array, crRNA, gRNA, engineeredsequence or vector is for in vitro treating an industrial or medicalfluid, solid surface, apparatus or container (eg, for food, consumergoods, cosmetics, personal healthcare product, petroleum or oilproduction); or for treating a waterway, water, a beverage, a foodstuffor a cosmetic, wherein the host cell(s) are comprised by or on thefluid, surface, apparatus, container, waterway, water, beverage,foodstuff or cosmetic.

The invention also provides: An ex vivo mixed population of bacteriaobtainable by the use or method of any concept herein.

In an example, the mixed population or the product of the use or methodis in a container for medical or nutritional use. For example, thecontainer is a sterilised container, eg, an inhaler or connected to asyringe or IV needle.

In an example, the product population of the use or method is useful foradministration to a human or animal to populate a microbiome thereof.

The invention provides: A foodstuff or beverage for human or non-humananimal consumption comprising the population product of the use ormethod.

Herein, in an example of any configuration, concept or aspect, theBacteroides is a species selected from caccae, capillosus,cellulosilyticus, coprocola, coprophilus, coprosuis, distasonis, dorei,eggerthii, faecis, finegoldii, fluxus, fragalis, intestinalis,melaninogenicus, nordii, oleiciplenus, oralis, ovatus, pectinophilus,plebeius, stercoris, thetaiotaomicron, uniformis, vulgatus andxylanisolvens. For example, the Bacteroides is thetaiotaomicron, eg,wherein the host cell or mixed population is a gut microbiota populationex vivo or in vitro. In an example, the host cells, first or secondbacteria sub-population comprises a plurality of different Bacteroidetesspecies, or a plurality of Bacteroides species (eg, comprising Bthetaiotaomicron and B fragalis), or Bacteroides and Prevotella species.Herein, in an example, the Prevotella is a species selected frombergensis, bivia, buccae, buccalis, copri, melaninogenica, oris,ruminicola, tannerae, timonensis and veroralis. In an alternative, thehost cells, first or second bacteria are Firmicutes cells. In anexample, the host cells, first or second sub-population comprises orconsists of one or more Firmicutes selected from Anaerotruncus,Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerococcus,Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio,Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea, Enterococcus,Ethanoligenens, Faecalibacterium, Fusobacterium, Gracilibacter,Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus,Leuconostoc, Megamonas, Moryella, Mitsuokella, Oribacterium, Oxobacter,Papillibacter, Proprionispira, Pseudobutyrivibrio, Pseudoramibacter,Roseburia, Ruminococcus, Sarcina, Seinonella, Shuttleworthia,Sporobacter, Sporobacterium, Streptococcus, Subdoligranulum,Syntrophococcus, Thermobacillus, Turibacter and Weisella. In an example,the host cells, or the first or second sub-population consists ofClostridium cells (and optionally the other sub-population consists ofBacteroides (eg, thetaiotaomicron) cells). In an example, the hostcells, or the first or second sub-population consists of Enterococcuscells (and optionally the other sub-population consists of Bacteroides(eg, thetaiotaomicron) cells). In an example, the host cells, or thefirst or second sub-population consists of Ruminococcus cells (andoptionally the other sub-population consists of Bacteroides (eg,thetaiotaomicron) cells). In an example, the host cells, or the first orsecond sub-population consists of Streptococcus cells (and optionallythe other sub-population consists of Bacteroides (eg, thetaiotaomicron)cells). In an example, the host cells, or the first or secondsub-population consists of Faecalibacterium cells (and optionally theother sub-population consists of Bacteroides (eg, thetaiotaomicron)cells). For example, the Faecalibacterium is a Faecalibacteriumprausnitzii (eg, A2-165, L2-6, M21/2 or SL3/3).

In an example, the host cells, or the first or second sub-populationcomprises or consists of one or more Firmicutes selected fromAnaerotruncus, Acetanaerobacterium, Acetitomaculum, Acetivibrio,Anaerococcus, Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax,Butyrivibrio, Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea,Enterococcus, Ethanoligenens, Faecalibacterium, Fusobacterium,Gracilibacter, Guggenheimella, Hespellia, Lachnobacterium, Lachnospira,Lactobacillus, Leuconostoc, Megamonas, Moryella, Mitsuokella,Oribacterium, Oxobacter, Papillibacter, Proprionispira,Pseudobutyrivibrio, Pseudoramibacter, Roseburia, Ruminococcus, Sarcina,Seinonella, Shuttleworthia, Sporobacter, Sporobacterium, Streptococcus,Subdoligranulum, Syntrophococcus, Thermobacillus, Turibacter andWeisella. In an example, the host cells, or the first or secondsub-population consists of Clostridium (eg, dificile) cells (andoptionally the other sub-population consists of Bacteroides (eg,thetaiotaomicron) cells). In an example, the host cells, or the first orsecond sub-population consists of Enterococcus cells (and optionally theother sub-population consists of Bacteroides (eg, thetaiotaomicron)cells). In an example, the host cells, or the first or secondsub-population consists of Ruminococcus cells (and optionally the othersub-population consists of Bacteroides (eg, thetaiotaomicron) cells). Inan example, the host cells, or the first or second sub-populationconsists of Streptococcus cells (and optionally the other sub-populationconsists of Bacteroides (eg, thetaiotaomicron) and/or Enterobacteriaceae(eg, E coli) cells). In an example, the host cells, or the first orsecond sub-population consists of Faecalibacterium cells (and optionallythe other sub-population consists of Bacteroides (eg, thetaiotaomicron)cells). In an example, the host cells, or the first or secondsub-population consists of Streptococcus cells (optionally Sthermophilus and/or pyogenes cells) and the other sub-populationconsists of Bacteroides (eg, thetaiotaomicron) and/or Enterobacteriaceae(eg, E coli) cells.

The population product of the use or method of the invention is, in anembodiment, for administration to a human or non-human animal bymucosal, gut, oral, intranasal, intrarectal, intravaginal, ocular orbuccal administration.

Optionally the host cells, or the first or second sub-populationbacteria are B fragalis bacteria and the population is harboured bywater.

A suitable beverage comprising an array, system, engineered sequence,vector or gRNA of the invention is, for example, a probiotic drink, eg,an adapted Yakult (trademark), Actimel (trademark), Kevita (trademark),Activia (trademark), Jarrow (trademark) or similar drink for humanconsumption.

Phage Sequence Targets

In aspects of the invention, the target sequence is a sequence of aphage that infects a host bacterial cell. Desired modification of phagegenomes, as achieved by the invention, not only relates to phage killingor knock-down, but instead can be desired phage gene or regulatoryelement activation in the host cell (eg, when the phage expresses adesired protein or other product that is associated with increased hostcell viability or proliferation). Alternatively, modification may beinducible phage gene expression regulation, eg, by use of an inducibleCas that is targeted according to the invention to the phage targetsite. In an embodiment, the invention provides for modifying the phagetarget site by cutting with a Cas nuclease in the host cell. This may beuseful for various reasons, for example:—

A. to mutate the target site to activate or inactivate it (eg, for geneknock-down or inactivation of an anti-host gene; or for killing the hostcell when the phage target is integrated in the host chromosome);B. to delete the target sequence or a larger sequence comprising thetarget sequence (eg, when the invention is used with first and secondPM-crRNAs that target spaced sites in the phage genome, wherein cuts ineach site result in deletion of phage nucleic acid between the cuts);C. to insert a desired PM-DNA sequence into the host cell genome (eg, byproviding one or more PM-crNA-guided cuts in a host nucleic acid forhomologous recombination insertion of the desired PM-DNA).

The invention provides the following aspects:—

1. A method of altering the relative ratio of sub-populations of firstand second bacteria in a mixed population of bacteria comprising saidsub-populations, wherein the first bacteria are host cells (eg,Bacteroidetes host cells) (wherein the first bacteria are optionallyinfected by a phage and the second bacteria are not infected by saidphage (or not Bacteroidetes)), the method comprising combining the mixedpopulation with a plurality of vectors in one or more steps forintroduction of vector nucleic acid (eg, a PM-containing transposonthereof) into host cells and allowing bacterial growth in the mixedpopulation, wherein the relative ratios of said first and secondbacteria is altered;

wherein each vector comprises an engineered phage-modifying (PM) CRISPRarray for introduction into host cell for modifying a target nucleotidesequence (eg, of said phage) in the cell,(a) wherein the PM-CRISPR array comprises one or more sequences forexpression of a PM-crRNA respectively and a promoter for transcriptionof the sequence(s) in a host cell; and(b) wherein the PM-crRNA is capable of hybridising to the targetsequence to guide Cas (eg, a Cas nuclease) in the host cell to modifythe target sequence.

By targeting phage sequence(s) to inactivate gene(s) required for phageviability, propogation or infectivity, in one aspect the inventionprovides the array with a positive selective advantage that may promoteits uptake and retention by host cells infected with the phage. Whenhost cells are killed or growth is reduced, the relative ratio of firstto second bacteria in the population is reduced. The invention providessuch a product population, eg, for use as a medicament for treatment orprevention (reducing the risk) of a disease or condition in a human oranimal subject, wherein the medicament is administered to the subject.The disease or condition can be any disease or condition disclosedherein. In an example, a single guide RNA (gRNA) is expressed in thehost cells to provide the crRNA and each vector comprises an expressibleengineered nucleotide sequence encoding such a gRNA.

In an example using a PM-array, the target sequence is a Bacteroidesthetaiotaomicron sequence. Optionally the target sequence is notcomprised by B fragalis. This is useful, for example, where themodifying cuts or otherwise renders the target sequence non-functional,whereby the ratio of B thetaiotaomicron host cells is increased withouttargeting B fragalis, eg, where the mixed population is a gut microbiotapopulation as described herein. B fragalis is in some settingsassociated with abscesses and thus this example reduces the risk ofthis, whilst enabling alteration of ratios (increase of Bthetaiotaomicron cell proportion) as per the invention that is usefulfor example to re-balance gut microbiota, eg, for treating or preventingobesity or diabetes or IBD.

The promoter (or a HM- or PM-array) is operable in a host cell. In anexample, the promoter is a viral or phage promoter, eg, a T7 promoter.In another example, the promoter is a bacterial promoter (eg, a promoterof the host cell species).

2. The method of aspect 1, wherein the first bacteria are Bacteroides(eg, thetaiotamicron or fragalis), Alistipes, Alkaliflexus,Parabacteroides, Tannerella, Xylanibacter and/or Prevotella bacteria.

3. The method of aspect 1 or 2, wherein the second bacteria areFirmicutes bacteria (eg, when the first bacteria are Bacteroidetes orBacteroides).

4. The method of any preceding aspect, wherein the ratio of the firstbacteria sub-population to the second bacteria sub-population isincreased, ie, is greater after said method has been carried out thanbefore.

5. The method of aspect 4, wherein the mixed population is comprised bya composition (eg, a beverage, mouthwash or foodstuff) foradministration to a human or non-human animal for populating andrebalancing the gut or oral microbiota thereof, eg, wherein the mixedpopulation is in vitro, or in vivo in the human or non-human animal. Themethod of aspect 1, 2 or 3, wherein the ratio of the first bacteriasub-population to the second bacteria sub-population is decreased, ie,is less after said method has been carried out than before.

6. The method of aspect 6, wherein the mixed population is harboured bya beverage or water (eg, a waterway or drinking water) for humanconsumption.

7. The method of any preceding aspect, wherein each vector is a plasmid,phage (eg, a packaged phage) or phagemid.

8. The method of aspect 8, wherein each vector is a phage (eg, apackaged phage) and vector nucleic acid is introduced into host cells byphage vector nucleic acid transduction into host cells, ie, by infectionof host cells by phage vectors. In an example, the phage comprises oneor more transposons as described herein.

9. The method of aspect 8, wherein each vector is a plasmid and vectornucleic acid is introduced into host cells by transformation orhorizontal plasmid transfer from bacteria harbouring the vectors. In anexample, the plasmid comprises one or more transposons as describedherein. In an example, the bacteria harbouring the vectors is anon-Bacteroidetes or non-Bacteroides species.

Additionally or alternatively, the bacteria harbouring the vectors is anon-Firmicutes species. In an example, the bacteria harbouring thevectors are bacteria of one or more species selected from the groupconsisting of a Lactobacillus species (eg, acidophilus (eg, La-5, La-14or NCFM), brevis, bulgaricus, plantarum, rhammosus, fermentum,caucasicus, helveticus, lactis, reuteri or casei eg, casei Shirota), aBifidobacterium species (eg, bifidum, breve, longum or infantis),Streptococcus thermophilus and Enterococcus faecium. For example, thebacteria are L acidophilus or lactis bacteria.

10. An engineered Bacteroidetes phage-modifying (PM) CRISPR array foruse in the method of any preceding aspect for modifying the genome ofsaid Bacteroidetes phage,

-   -   (a) wherein the PM-CRISPR array comprises one or more sequences        for expression of a PM-crRNA and a promoter for transcription of        the sequence(s) in a Bacteroidetes phage-infected host cell; and    -   (b) wherein the PM-crRNA is capable of hybridising to a        Bacteroidetes phage genome target sequence to guide Cas (eg, a        Cas nuclease) in the infected host cell to modify the target        sequence.

11. A nucleic acid vector (eg, a plasmid, phage or phagemid) for use inthe method of any one of aspects 1 to 10, the vector comprising aPM-CRISPR array of aspect 11.

In a general embodiment of the invention, there is alternativelyprovided for aspect 12:—

A nucleic acid vector (eg, a plasmid, virus, phage or phagemid)comprising an engineered HM-CRISPR array for modifying a target sequenceof the genome of a host bacterial cell (eg, pathogenic bacterial cell,such as described above) or the genome of a virus (eg, phage) in a hostcell,

(a) wherein the CRISPR array comprises one or more sequences forexpression of a crRNA and a promoter for transcription of thesequence(s) in the host cell; and(b) wherein the crRNA is capable of hybridising to the target sequenceto guide Cas (eg, a Cas nuclease) in the host cell to modify the targetsequence.

The promoter is operable in a host cell. In an example, the promoter isa viral or phage promoter, eg, a T7 promoter. In another example, thepromoter is a bacterial promoter (eg, a promoter of the host cellspecies).

In an example, the array is comprised by a transposon described herein.In an example, the array is comprised by a carrier bacterium asdescribed herein. In an example, a plurality of the arrays is providedfor targeting one or more target nucleotide sequences of the phage orhost cell, wherein the plurality of arrays are comprised by bacterialcells, eg, carrier, first recipient or second recipient cells asdescribed herein. In an example, the carrier cells are comprised by abeverage (eg, a probiotic drink for human consumption) or foodstuff asdescribed herein. In an example, the array or carrier bacteria are foradministration to a human or non-human animal for treating or preventingan infection of the human or animal, eg wherein the host cell ispathogenic. In an example, the array or carrier bacteria are foradministration to the gut of a human or non-human animal for treating orpreventing obesity, diabetes or IBD of the human or animal.

12. The array or vector of aspect 11 or 12 wherein the array or vectoris comprised by a bacterial cell, eg, a probiotic cell for human ornon-human animal consumption.

13. The method, array or vector of any preceding aspect, wherein thevectors are comprised by a third bacterial population (eg, carrierbacteria described herein) that is used for said combining with themixed population or is for combination with the mixed population,whereby vector nucleic acid is introduced into host cells bytransformation (eg, by horizontal plasmid vector or transposon transferfrom the third bacteria to the first bacteria host cells) ortransduction (eg, by phage vector infection of first bacteria hostcells).

14. The method, array or vector of any preceding aspect, wherein the oreach array or vector is comprised by a human or non-human animal gutcommensal or symbiotic bacterial cell (eg, a carrier bacterial cell asdescribed herein). Thus, the cell is of a gut bacterial species that iscommensal or symbiotic with the human or non-human animal.

15. The method or vector of any one of aspects 12 to 15, wherein the oreach vector is a plasmid, phage or phagemid comprising an origin ofreplication that is operable in a Firmicutes host cell or in aBacteroidetes phage-infected host cell (eg, a Bacteroides cell), andoptionally operable in a commensal or symbiotic bacterial cell asdefined in aspect 15. In an example, the origin of replication is oriTor any other origin of replication described herein.

16. The method or vector of any one of aspects 12 to 16, wherein the oreach vector is a plasmid or phagemid comprising a sequence (eg, atransposon described herein) that is capable of horizontal transferbetween (1) a human or non-human animal commensal or symbiotic bacterialcell that is not a Bacteroides cell and (2) a said phage-infected cellwhich is a Bacteroides cell; or between (3) a human or non-human animalcommensal or symbiotic bacterial cell that is not a Firmicutes cell and(4) a Firmicutes cell comprising the target sequence.

17. The method or vector of any one of aspects 12 to 17, wherein the oreach vector is a plasmid or phagemid sequence (eg, a transposondescribed herein) that is capable of horizontal transfer between (1) asaid phage-infected cell which is a Bacteroides cell and (2) a bacterialcell that is suitable for probiotic administration to a human ornon-human animal gut; or between (3) a Firmicutes cell comprising thetarget sequence and (4) a bacterial cell that is suitable for probioticadministration to a human or non-human animal gut.

18. The method or vector of any one of aspects 15 to 18, wherein thecommensal, symbiotic or probiotic species is selected from the groupconsisting of a Lactobacillus species (eg, acidophilus (eg, La-5, La-14or NCFM), brevis, bulgaricus, plantarum, rhammosus, fermentum,caucasicus, helveticus, lactis, reuteri or casei eg, casei Shirota), aBifidobacterium species (eg, bifidum, breve, longum or infantis),Streptococcus thermophilus and Enterococcus faecium.

The method, array or vector of any preceding aspect, wherein thepromoter is operable for transcription of said sequence(s) in a saidphage-infected Bacteroidetes host cell and in a commensal, symbiotic orprobiotic bacterial cell as defined in any one of aspects 15 to 19; orin a Firmicutes cell comprising the target sequence and in a commensal,symbiotic or probiotic bacterial cell as defined in any one of aspects15 to 19. For example, the promoter is a viral or bacterial promoter,eg, a T7 promoter. In an example, the promoter is a host cell promoter,eg, a promoter of a host CRISPR/Cas array.

19. The method, array or vector of any preceding aspect, or any useherein, wherein the modifying is (i) cutting of the target sequence,(ii) downregulating transcription of a gene comprising the targetsequence, (iii) upregulating transcription of a gene comprising thetarget sequence, or (iv) adding, deleting or substituting a nucleic acidsequence at the target.

20. The method, array or vector of any preceding aspect, wherein theBacteroidetes phage is a Bacteroides phage selected from a crAssphage, aGB-124 phage, a GA-17 phage, a HB-13 phage, a H16-10 phage, a B40-8phage and B fragalis phage ATCC51477-B1. Reference is made to NatCommun. 2014 Jul. 24; 5:4498. doi: 10.1038/ncomms5498, “A highlyabundant bacteriophage discovered in the unknown sequences of humanfaecal metagenomes”, Dutilh B E et al. The crAssphage ˜97 kbp genome issix times more abundant in publicly available metagenomes than all otherknown phages together; it comprises up to 90% and 22% of all reads invirus-like particle (VLP)-derived metagenomes and total communitymetagenomes, respectively; and it totals 1.68% of all human faecalmetagenomic sequencing reads in the public databases. Using a newco-occurrence profiling approach, Dutilh et al predicted a Bacteroideshost for this phage, consistent with Bacteroides-related proteinhomologues and a unique carbohydrate-binding domain encoded in the phagegenome.

21. The method, array or vector of any preceding aspect, or any useherein, wherein the target sequence is comprised by a phage generequired for host cell infectivity, the phage lysogenic or lytic cycle,or phage viability, eg, an essential gene or coat protein gene.

22. The method, array or vector of any preceding aspect, wherein thetarget sequence is comprised by a BACON (Bacteroidetes-associatedcarbohydrate-binding) domain-encoding sequence (eg, wherein the host isa Bacteroides host) or an endolysin-encoding sequence. Reference is madeto FEBS Lett. 2010 Jun. 3; 584(11):2421-6, doi:10.1016/j.febslet.2010.04.045. Epub 2010 Apr. 21, “Mining metagenomicdata for novel domains: BACON, a new carbohydrate-binding module”, MelloL et al. The presence of the BACON domain in a phage-structural proteinmight be explained by the proposed bacteriophage adherence to mucusmodel. According to this model, phage adhere to the mucin glycoproteinscomposing the intestinal mucus layer through capsid-displayedcarbohydrate-binding domains (such as the immunoglobulin-like fold orthe BACON domain), facilitating more frequent interactions with thebacteria that the phage infects.

25. The method, array or vector of any preceding aspect, or any useherein, wherein the CRISPR array comprises a sequence R1-S1-R1′ forexpression and production of the crRNA in the host cell,

(i) wherein R1 is a first CRISPR repeat, R1′ is a second CRISPR repeat,and R1 or R1′ is optional; and(ii) S1 is a first CRISPR spacer that comprises or consists of anucleotide sequence that is 95% or more identical to said targetsequence. For example, the target sequence comprises a protospacer or iscomprised by a protospacer sequence that is immediately adjacent to aprotospacer adjacent motif (PAM) that is cognate to a Cas when the arrayof the invention is in the host cell, wherein the Cas is also cognate tothe crRNA expressed from the array. In an embodiment, the Cas isendogenous to the cell. In another example, the Cas is exogenous to thehost cell, eg, provided by a vector of the invention.

26. The method, array or vector of aspect 25, wherein R1 and R1′ are atleast 95% (eg, 96, 97, 98, 99 or 100%) identical to repeat sequences ofa CRISPR array of a cell of the same species as the host cell.

27. The method, array or vector of aspect 25, wherein R1 and R1′ is eachat least 95% (eg, 96, 97, 98, 99 or 100%) identical to a repeat sequenceof a CRISPR array (eg, a Type II-C array) of a Bacteroides speciesselected from thetaiotamicron and fragalis (eg, Bacteroides fragalisNCTC 9343), wherein the host cells comprise a CRISPR/Cas system that isfunctional with the repeat sequence and are Bacteroides cells, eg, ofsaid species.

28. The method, array, use or vector of aspect 27, wherein R1 and R1′are at least 95% (eg, 96, 97, 98, 99 or 100%) identical respectively tothe first (5′-most) and second (the repeat immediately 3′ of the firstrepeat) repeat sequences of a CRISPR array of said species, eg, of asaid host cell of said species. In an example, the array is a Type II-Carray. In an example, the array or vector further comprises R2-S2-R2′,wherein the spacer S2 is the same or different from the spacer S1 (eg,for targeting a different target site in the host cell or phage genome),wherein R2 and R2′ are functional in the host cell and are optionallythe same as R1. For example, each of R1, R1′, R2 and R2′ is a B fragalisCRISPR repeat.

29. The method, array, use or vector of aspect 25, wherein (iii) each ofR1 and R1′ is identical to a repeat sequence of a CRISPR array (eg, aType II-C array) of a Bacteroides species cell, wherein the species isselected from the group consisting of caccae, capillosus,cellulosilyticus, coprocola, coprophilus, coprosuis, distasonis, dorei,eggerthii, faecis, finegoldii, fluxus, fragalis (eg, fragalis NCTC9343), intestinalis, melaninogenicus, nordii, oleiciplenus, oralis,ovatus, pectinophilus, plebeius, stercoris, thetaiotaomicron, uniformis,vulgatus and xylanisolvens, and (iv) wherein the host cell comprises aCRISPR/Cas system that is functional with the repeat sequence and is aBacteroides cell of a species selected from said group (eg, the samespecies as the selected species of (iii)).

30. The method, array, use or vector of aspect 25, wherein R1 and R1′are functional with a CRISPR/Cas system of a said host Bacteroidetes orFirmicutes cell for modification of the target sequence. In an example,R1, R1′, R2 and R2′ are Type II (eg, Type II-C) CRISPR/Cas systemrepeats of the same bacterial species, eg, a Bacteroides, such asthetaiotamicron or fragalis or Streptococcus, such as thermophilusorpyogenes.

31. The method, array, use or vector of aspect 25, wherein R1 and R1′are at least 95% (eg, 96, 97, 98, 99 or 100%) identical to repeatsequences of a CRISPR array (eg, a Type II-C array) of a Bacteroidetes(eg, Bacteroides or Prevotella) or Firmicutes (eg, Streptococcus) cell.

32. The method, array, use or vector of aspect 25, wherein each of R1and R1′ is at least 95% (eg, 96, 97, 98, 99 or 100%) identical to asequence selected from SEQ ID NOs: 1 to 5 of Table 2 and optionally thefirst bacterial cells are Bacteroides cells, eg, of a species or strain(eg, the species or strain listed against the selected sequence) inTable 2.

33. The method, array, use or vector of aspect 25, wherein each of R1and R1′ is at least 95% (eg, 96, 97, 98, 99 or 100%) identical to asequence selected from SEQ ID NOs: 6 to 11 Table 2 of and optionally thefirst bacterial cells are Prevotella cells, eg, of a species or strain(eg, the species or strain listed against the selected sequence) inTable 2.

34. The method, array or vector of any preceding aspect, wherein the oreach array is in combination with one or more Cas nuclease(s) thatfunction with the crRNA in a said host cell to modify the targetsequence. For example, the target sequence comprises a protospacersequence immediately adjacent to a Protospacer Adjacent Motif (PAM),optionally wherein the PAM is cognate to a Cas nuclease comprised by theBacteroidetes host cells. In an example, the Cas is a Type II-C Casnuclease.

35. The method, array or vector of any preceding aspect, wherein the oreach array is in combination with nucleic acid sequence(s) encoding oneor more Cas nuclease(s) that function with the crRNA in a said host cellto modify the target sequence.

36. The method, array, use or vector of aspect 25, wherein R1 and R1′are functional with a Type II Cas9 nuclease (eg, a S pyogenes, Sthermophilus or S aureus Cas9) to modify the target in a said host cell,optionally wherein the method, array or vector is further according toaspect 34 or 35 wherein the Cas is said Cas9.

37. An ex-vivo mixed population of bacteria obtainable by the method ofany one of aspects 1 to 10 or 14 to 36 or a use herein. For example, themixed population is in a container for medical or nutritional use. Forexample, the container is a sterilised container.

38. A composition for administration to a human or non-human animal fortherapeutic, prophylactic, cosmetic, human or non-human animal body massreduction (eg, cosmetic reduction) or nutritional use, the compositioncomprising the mixed population of aspect 37. In an example, thecomposition is for oral, systemic, inhaled, intrarectal, ocular, buccalor intravaginal administration. In an example, the composition is foradministration to the gut or oral cavity of a human or non-human animal.

39. A foodstuff or beverage for human or non-human animal consumptioncomprising the mixed population of aspect 37 or the composition ofaspect 38.

40. The foodstuff or beverage of aspect 39, which is a nutritionalsupplement or a probiotic beverage or foodstuff.

41. An antibiotic composition for treating or preventing a Bacteroidetesinfection in a human or non-human animal or in drinking water, whereinthe composition comprises an array or vector of any one of aspects 11 to36, optionally wherein the modifying is according to aspect 21 (iii) or(iv).

42. A probiotic composition for increasing the proportion of gutBacteroidetes (eg, to treat or prevent obesity, diabetes (eg, Type I) ora GI inflammatory condition) in a human or non-human animal, wherein thecomposition comprises an array or vector of any one of aspects 11 to 36,optionally wherein the modifying is according to aspect 21 (iii) or(iv).

43. The composition of aspect 38, 41 or 42 for increasing the relativeproportions of gut Bacteroides to Firmicutes in the human or animal, egfor treating or preventing obesity, diabetes (eg, Type I diabetes) or aGI condition (eg, Crohn's disease, IBD, IBS or ulcerative colitis).

In an alternative, “array” in any configuration of the invention caninstead by an engineered nucleotide sequence encoding a HM-crRNA or gRNAfor expression in a host cell. The features of any of the aspects hereinrelating to an array can, therefore, in the alternative apply mutatismutandis to such an engineered sequence.

Mobile Genetic Elements & CRISPR Systems

44. A nucleic acid vector (eg, a plasmid, virus, phage or phagemid)comprising an engineered CRISPR array for modifying a target sequence ofthe genome of a host bacterial cell (eg, Firmicutes or pathogenicbacterial cell, such as described above) or the genome of a virus (eg,phage) in a host cell,

(a) wherein the CRISPR array comprises one or more sequences forexpression of a crRNA (eg, comprised by a gRNA) and a promoter fortranscription of the sequence(s) in the host cell;(b) wherein the crRNA is capable of hybridising to the target sequenceto guide Cas (eg, a Cas nuclease) in the host cell to modify the targetsequence;(c) wherein the array is comprised by a transposon that is capable ofhorizontal transfer between first and second bacterial cells ofdifferent species.

Optionally, the Cas nuclease is a wild-type endogenous Cas nuclease ofthe host cell.

45. The vector of aspect 44, wherein the array is for administration toa human or non-human animal; and the first cell species isnon-pathogenic to the human or animal and the second cell species ispathogenic to the human or animal, wherein the array is comprised by thefirst cell.

46. The vector of aspect 45, wherein the first cell species is a speciesthat is commensal or symbiotic with the human or animal, eg, a gutmicrobiota species.

47. The vector of aspect 45 or 46, wherein the first cell species isselected from the group consisting of a Lactobacillus species (eg,acidophilus (eg, La-5, La-14 or NCFM), brevis, bulgaricus, plantarum,rhammosus, fermentum, caucasicus, helveticus, lactis, reuteri or caseieg, casei Shirota), a Bifidobacterium species (eg, bifidum, breve,longum or infantis), Streptococcus thermophilus and Enterococcusfaecium.

48. The vector of any one of aspects 44 to 47, wherein the vector iscomprised by a beverage (eg, a probiotic drink) or foodstuff for humanor animal consumption.

49. The vector of any one of aspects 44 to 48, wherein the vectorcomprises at least one repeat-spacer-repeat unit for targeting thetarget sequence, wherein the repeats are at least 95% (eg, 96, 97, 98,99 or 100%) identical to repeats of a CRISPR/Cas system of the hostcell, whereby the repeats of the vector are operable in the host cell toguide Cas of the host system to modify the target nucleotide sequence.

50. The vector of aspect 49, wherein the vector lacks a Cas (eg, Casnuclease)-encoding sequence.

Targeting of a nucleotide sequence of the host CRISPR/Cas systemaccording to the invention is useful for removing host cell resistanceto a vector (eg, invading virus) or reducing the development or increaseof resistance. For example, the invention thereby provides the advantageof targeting and knocking down the activity of an endogenous CRISPR/Cassystem so that new vector (eg, phage) spacer acquisition is inhibited.

A feature of mobilisation is the presence of a cis-acting region (oriT)that is required for transfer. This region is the initiation site of DNAprocessing at which a site- and strand-specific nick is made in theplasmid to start the transfer event. The invention provides furtherembodiments employing mobile genetic elements (MGEs) as follows:—

1. An engineered CRISPR nucleic acid vector comprising or consisting ofa mobile genetic element (MGE), wherein the MGE comprises an origin oftransfer (oriT) and a CRISPR array for modifying a target sequence ofthe genome of a host cell (eg, pathogenic bacterial cell) or the genomeof a virus (eg, prophage) in a host cell,

(a) wherein the CRISPR array comprises one or more sequences forexpression of a crRNA and a promoter for transcription of thesequence(s) in the host cell;(b) wherein the crRNA is capable of hybridising to the target sequenceto guide Cas (eg, a Cas nuclease) in the host cell to modify the targetsequence;(c) wherein the vector is capable of transfer between (i) first andsecond nucleic acid positions of a first host cell, wherein eachposition is a position on a chromosome or a plasmid and the targetsequence is comprised by the host cell, or (ii) first and second hostcells, wherein the target sequence is comprised by the first and/orsecond host cell.

Examples of MGEs are ICEs, transposons, plasmids and bacteriophage. Anorigin of transfer (oriT) is a short sequence (eg, up to 500 bp) that isnecessary for transfer of the DNA that contains it from a bacterial hostto recipient during conjugation. The oriT is cis-acting—it is found onthe same DNA that is being transferred, and it is transferred along withthe DNA. A typical origin of transfer comprises three functionallydefined domains: a nicking domain, a transfer domain, and a terminationdomain.

Optionally, the promoter is operable for transcription of saidsequence(s) in the first and second (and optionally the third) cells.

Optionally the target sequence is comprised by the second cell.Optionally the target sequence is not comprised by the second cell.

In an example, the first and second cells are of different bacterialspecies (eg, species found in a human microbiome population, eg, of thegut, armpit, vagina or mouth). In an example, the first and second cellsare ex vivo. In another example, the first and second cells arecomprised by a human gut, vaginal, armpit or oral microbiome in vivo orex vivo.

2. The vector of embodiment 1, wherein the MGE is or comprises anintegrative and conjugative element (ICE). Alternatively, the MGE is amobilisable MGE (ie, able to use factors encoded by genes not carried bythe MGE, in order to be mobilised). The terms “mobilisable” and“conjugative” in relation to MGEs are readily apparent to the skilledaddressee.

Reference is made to the ICEberg database(http://db-mml.sjtu.edu.cn/ICEberg/), which provides examples ofsuitable ICEs for the invention and sources for suitable oriT. In anexample, the ICE is a member of an ICE family comprising an ICE selectedfrom the group 1 to 28, or the oriT is an oriT of a member of such afamily: 1=SXT/R391; 2=Tn916; 3=Tn4371; 4=CTnDOT/ERL; 5=ICEc1c; 6=ICEBs1;7=ICEHin1056; 8=PAPI-1; 9=ICEM1Sym(R7A); 10=ICESt1; 11=SPI-7;12=ICE6013; 13=ICEKp1; 14=TnGBS1; 15=Tn5253; 16=ICESa2603; 17=ICEYe1;18=10270-RD.2; 19=Tn1207.3; 20=Tn1806; 21=ICEA5632; 22=ICEF-I/II;23=ICEAPG2; 24=ICEM; 25=10270-RD.1; 26=Tn5801; 27=PPI-1; 28=ICEF-III.Family descriptions are found in the ICEberg database. For example, theTn916 family was defined by Roberts et al (2009) (Trends Microbiol. 2009June; 17(6):251-8. doi: 10.1016/j.tim.2009.03.002. Epub 2009 May 20; “Amodular master on the move: the Tn916 family of mobile geneticelements”, Roberts A, Mullany P). Elements belonging to the Tn916 familyare defined by the following criteria: they must have the generalorganization shown in Roberts et al, and they must have a core region(conjugation and regulation module) that is similar in sequence andstructure to the original Tn916 at the DNA level. Exceptions are someconjugative transposons, such as Tn1549 which have been previouslyclassified in this family and those with a high degree of proteinsimilarity as described in corresponding references.

3. The vector of embodiment 2, wherein the ICE is a transposon, eg, aconjugative transposon. In an example, the MGE is a mobilisabletransposon that is mobilisable in the presence of a functional helperelement, optionally wherein the transposon is in combination with a saidhelper element.

4. The vector of any preceding embodiment, wherein the vector is aplasmid, optionally wherein the MGE is a transposon comprised by theplasmid. For example, the transposon is a conjugative transposon. In anexample the transposon is a mobilisable transposon (eg, mobilisableusing one or more factors encoded by the plasmid, eg, by genes outsidethe transposon sequence of the plasmid). Optionally, the transposon is aType I transposon. Optionally, the transposon is a Type II transposon.

5. The vector of any preceding embodiment, wherein oriT is functional inthe first and second host cells. This is useful to promote spread andpropogation across bacteria in a bacterial population, eg, when thefirst and second cells are of different species.

6. The vector of embodiment 5 when comprised by the first cell, whereinthe first cell comprises nucleotide sequences encoding proteins operableto transfer the MGE to the second cell, wherein the sequences are notcomprised by the MGE. This is useful to avoid using space in the MGE forsuch sequences. For example, this enables construction of a more compactMGE for transfer between cells or enables inclusion of larger or moreCRISPR arrays, eg, to include a plurality of spacers to targetrespective sequences in a host cell or to target different sequences inthe first and second host cells.

7. The vector of embodiment 6, wherein the sequences are not comprisedby the vector. This is useful to avoid using space in the vector or MGEfor such sequences. For example, this enables construction of a morecompact vector or MGE for transfer between cells or enables inclusion oflarger or more CRISPR arrays, eg, to include a plurality of spacers totarget respective sequences in a host cell or to target differentsequences in the first and second host cells, and/or to include one ormore sequences for encoding Cas protein(s), eg a Cas9.

8. The vector of embodiment 6 or 7, wherein the sequences are comprisedby a conjugative transposon of the first cell. This is useful since itenables harnessing of factors outside the MGE to effect conjugativetransposition, for horizontal transfer of the MGE of the inventionbetween first and second host cells (eg, of different bacterial speciesin a human microbiome).

9. The vector of embodiment 8, wherein the transposon is operable intrans to transfer the MGE to the second cell. This is useful since itenables harnessing of factors outside the MGE to effect conjugativetransposition, for horizontal transfer of the MGE of the inventionbetween first and second host cells (eg, of different bacterial speciesin a human micribiome). For example, the oriT of the MGE of theinvention is the same as an oriT comprised by a conjugative transposonof the host cell. This is useful to enable the MGE of the invention tooperate with factors encoded by the host cell for effecting horizontaltransfer of the MGE between the first and second host cells (eg,bacterial cells of different species, eg, human microbiome species).This enables the MGE to be more compact or frees up space for CRISPRarrays and/or Cas gene(s) as discussed above. The term “operable intrans” means that the MGE (ICE) is operable for horizontal transferusing proteins expressed from host nucleotide sequences outside thevector nucleotide sequences (eg, proteins expressed by a conjugativetransposon of the host cell) to transfer the MGE (or the entire vector,such as a plasmid containing the MGE) into the second cell.

10. The vector of any preceding embodiment when comprised by the firstcell, wherein the oriT of the MGE is the same as an oriT comprised by anICE of the first cell, wherein the ICE is operable in trans to transferthe MGE to the second cell.

11. The vector of any preceding embodiment, wherein the vector oriT isan oriT of a Bacteroidetes (eg, Bacteroidales or Bacteroides) orPrevotella transposon. This useful when the first and/or second hostcell is a Bacteroidetes (eg, Bacteroidales or Bacteroides) or Prevotellacell respectively. For example, the first cell is a cell of such aspecies and the second cell is a Firmicutes cell, the target sequencebeing comprised by the second cell but not the first cell, whereby theCRISPR array directs Cas in the second cell to cut the target sequence.In an example, the target sequence is comprised by an essential gene orantibiotic resistance gene of the second cell (and for the latter,optionally the vector is in combination with said antibiotic oradministered to a human or non-human animal in combination with saidantibiotic). Optionally, the transposon is a CTnDot or CTnERL transposonand the vector is in combination with tetracycline or administered to ahuman or non-human animal in combination with tetracycline.

12. The vector of any preceding embodiment, wherein the vector oriT is aCTnDot, CTnERL SXT/R391, Tn916 or Tn4371 family transposon oriT.

13. The vector of any preceding embodiment, wherein the MGE comprisesfirst and second terminal repeat sequences and the CRISPR array betweenthe repeat sequences.

14. The vector of any preceding embodiment, wherein the MGE leavesbehind a transposon copy (1) at the first nucleic acid position when ithas transferred to the second position; or (2) in the first cell whenthe it has transferred to the second cell. This is useful for promotingpropogation and maintenance of the MGE in a bacterial populationcomprising the host cell(s). In an alternative, the MGE does not leavebehind a transposon copy (i) at the first nucleic acid position when ithas transferred to the second position; or (ii) in the first cell whenthe it has transferred to the second cell.

15. The vector of any preceding embodiment when comprised by the firstand/or second cell (eg, first and second copies of the vector comprisedby the first and second cells).

16. The vector of embodiment 15, wherein the first and second cells arecells of different species. For example, the first cell is aLactobacillus cell (eg, as described herein) and/or the second cell is aBacteroidetes (eg, Bacteroides cell, eg, such a cell described herein)or a Firmicutes cell (eg, such a cell described herein). In an example,the first cell is a Bacteroidetes (eg, Bacteroides cell, eg, such a celldescribed herein) and the second cell is a Firmicutes cell (eg, such acell described herein), eg, for administration to a gut micribiome of ahuman for treating or preventing a GI condition or diabetes; or fortreating or preventing obesity.

17. The vector of embodiment 15 or 16, wherein the first and secondcells are bacterial or archaeal cells.

18. The vector of embodiment 16 or 17, wherein the first cell isnon-pathogenic in a human (eg, a commensal or symbiotic bacterial cell)and optionally the second cell is a pathogenic cell in a human. In analternative, the second cell is a non-pathogenic cell in a human. Theterm “non-pathogenic in a human” includes cells, such as certainbacterial species (eg, Bacteroides species, such as fragalis) that canreside in microbiomes of the human (eg, the gut, vaginal, armpit or oralmicrobiome) without pathogenicity or substantial pathogenicity, but inother environments of the human are pathogenic. The skilled person willreadily understand that the first cell type can be retained in or on ahuman and the second cell type should be reduced in or on the human. Forexample, the CRISPR array modifies the genome of the second cell to killor reduce cell viability or growth in or on the human. For example, thetarget site is comprised by the second cell and the site is cut by saidCas nuclease, thereby inactivating or down-regulating a gene comprisingthe target site. For example, the gene is an essential gene orantibiotic resistance gene of the second cell. In an example, the geneis a virulence gene.

19. The vector of any preceding embodiment, or any use herein, whereinthe second cell (each host cell) is a cell selected from (i) aStaphylococcus aureus cell, eg, resistant to an antibiotic selected frommethicillin, vancomycin-resistant and teicoplanin; (ii) a Pseudomonasaeuroginosa cell, eg, resistant to an antibiotic selected fromcephalosporins (eg, ceftazidime), carbapenems (eg, imipenem ormeropenem), fluoroquinolones, aminoglycosides (eg, gentamicin ortobramycin) and colistin; (iii) a Klebsiella (eg, pneumoniae) cell, eg,resistant to carbapenem; (iv) a Streptococcus (eg, pneumoniae orpyogenes) cell, eg, resistant to an antibiotic selected fromerythromycin, clindamycin, beta-lactam, macrolide, amoxicillin,azithromycin and penicillin; (v) a Salmonella (eg, serotype Typhi) cell,eg, resistant to an antibiotic selected from ceftriaxone, azithromycinand ciprofloxacin; (vi) a Shigella cell, eg, resistant to an antibioticselected from ciprofloxacin and azithromycin; (vii) a mycobacteriumtuberculosis cell, eg, resistant to an antibiotic selected fromResistance to isoniazid (INH), rifampicin (RMP), fluoroquinolone,amikacin, kanamycin and capreomycin; (viii) an Enterococcus cell, eg,resistant to vancomycin; (ix) an Enterobacteriaceae cell, eg, resistantto an antibiotic selected from a cephalosporin and carbapenem; (x) an E.coli cell, eg, resistant to an antibiotic selected from trimethoprim,itrofurantoin, cefalexin and amoxicillin; (xi) a Clostridium (eg,dificile) cell, eg, resistant to an antibiotic selected fromfluoroquinolone antibiotic and carbapenem; (xii) a Neisseria gonnorrhoeacell, eg, resistant to an antibiotic selected from cefixime (eg, an oralcephalosporin), ceftriaxone (an injectable cephalosporin), azithromycinand tetracycline; (xiii) an Acinetoebacter baumannii cell, eg, resistantto an antibiotic selected from beta-lactam, meropenem and a carbapenem;or (xiv) a Campylobacter cell, eg, resistant to an antibiotic selectedfrom ciprofloxacin and azithromycin. Such species can be pathogenic tohumans.

20. The vector or use of embodiment 19, wherein the target site iscomprised by an antibiotic resistance gene of the second cell, whereinthe antibiotic is a respective antibiotic recited in embodiment 19.

21. The vector of any one of embodiments 15 to 20, wherein the firstcell is a Bacteroidetes (eg, Bacteroidales or Bacteroides) cell;Lactobacillus (eg, acidophilus (eg, La-5, La-14 or NCFM), brevis,bulgaricus, plantarum, rhammosus, fermentum, caucasicus, helveticus,lactis, reuteri or casei eg, casei Shirota); Bifidobacterium (eg,bifidum, breve, longum or infantis); Streptococcus thermophiles;Enterococcus faecium; Alistipes; Alkaliflexus; Parabacteroides;Tannerella; or Xylanibacter cell.

22. The vector of any preceding embodiment, wherein the first and/orsecond nucleic acid positions of (i) are comprised by a Bacteroidetes(eg, Bacteroidales or Bacteroides) cell; or the first and/or second hostcells of (ii) are Bacteroidetes (eg, Bacteroidales or Bacteroides) orPrevotella cells.

23. The vector of embodiment 22, wherein the first cell is aBacteroidetes (eg, Bacteroidales or Bacteroides) cell and the secondcell is a Firmicutes (eg, Clostridium or Staphylococcus) cell, eg,wherein the vector is for administration to a gut micribiome of a humanfor treating or preventing a GI condition or diabetes; or for treatingor preventing obesity.

24. The vector of embodiment 16 or 17 (or any use herein), wherein thefirst cell (each first cell) is environmentally-acceptable in anenvironment (eg, in a water or soil environment) and optionally thesecond cell (each host cell) is not acceptable in the environment. Thewater environment will be readily apparent to the skilled person andcan, for example, be a marine or waterway (eg, lake, canal, river orreservoir) environment. In an example, the water environment is drinkingwater intended for human consumption or sewage water. In an example, thesoil environment is soil of farming land or soil at a mining site (eg, amineral or metal mining site).

By “acceptable” and “not acceptable” the skilled person will readilyunderstand that the first cell type can be retained in the environmentand the second cell type should be reduced in the environment. Forexample, the CRISPR array modifies the genome of the second cell to killor reduce cell viability or growth in the environment. For example, thetarget site is comprised by the second cell and the site is cut by saidCas nuclease, thereby inactivating or down-regulating a gene comprisingthe target site. For example, the gene is an essential gene orantibiotic resistance gene of the second cell. In an example, the geneis a virulence gene.

In an example, the environment is a microbiome of a human, eg, the oralcavity microbiome or gut microbiome or the bloodstream. In an example,the environment is not an environment in or on a human. In an example,the environment is not an environment in or on a non-human animal. In anembodiment, the environment is an air environment. In an embodiment, theenvironment is an agricultural environment. In an embodiment, theenvironment is an oil or petroleum recovery environment, eg, an oil orpetroleum field or well. In an example, the environment is anenvironment in or on a foodstuff or beverage for human or non-humananimal consumption.

In an example, the vector, system, vector, array, crRNA, gRNA, method orany use herein is for use in an industry or the environment is anindustrial environment, wherein the industry is an industry of a fieldselected from the group consisting of the medical and healthcare;pharmaceutical; human food; animal food; plant fertilizers; beverage;dairy; meat processing; agriculture; livestock farming; poultry farming;fish and shellfish farming; veterinary; oil; gas; petrochemical; watertreatment; sewage treatment; packaging; electronics and computer;personal healthcare and toiletries; cosmetics; dental; non-medicaldental; ophthalmic; non-medical ophthalmic; mineral mining andprocessing; metals mining and processing; quarrying; aviation;automotive; rail; shipping; space; environmental; soil treatment; pulpand paper; clothing manufacture; dyes; printing; adhesives; airtreatment; solvents; biodefence; vitamin supplements; cold storage;fibre retting and production; biotechnology; chemical; industrialcleaning products; domestic cleaning products; soaps and detergents;consumer products; forestry; fishing; leisure; recycling; plastics;hide, leather and suede; waste management; funeral and undertaking;fuel; building; energy; steel; and tobacco industry fields.

25. The vector of any preceding embodiment in combination with a nucleicacid (eg, a DNA) for incorporation at the modified target site.

In an example, the modification is cutting of the target site and thenucleic acid (eg DNA) is incorporated by homologous recombination in thehost cell. This is useful for effecting precise targeted modification ofthe host cell genome using the vector of the invention.

26. The vector of embodiment 25, wherein the nucleic acid forincorporation is or comprises a regulatory element or exon sequence, ega human sequence.

27. The vector of any preceding embodiment in combination with atransposase for mobilisation of the MGE.

28. The vector or any preceding embodiment, wherein the vector or MGEcomprises a toxin-antioxin module that is operable in the first hostcell; optionally wherein the toxin-antitoxin module comprises ananti-toxin gene that is not operable or has reduced operation in cellsother than the first cell.

29. The vector or any preceding embodiment, wherein the vector or MGEcomprises a toxin-antioxin module that is operable in the second hostcell; optionally wherein the toxin-antitoxin module comprises ananti-toxin gene that is not operable or has reduced operation in cellsother than the second cell.

30. The vector or any preceding embodiment, wherein the vector or MGEcomprises a toxin-antioxin module that is operable in the first andsecond host cells; optionally wherein the toxin-antitoxin modulecomprises an anti-toxin gene that is not operable or has reducedoperation in cells other than the first and second cells. The use of atoxin-antitoxin module is useful to confer selective advantages and thusMGE retention and spread. For example, the module is a Type I module,eg, a Hok-Sok module. For example, the module is a Type II module, eg, aHiCa-HicB module. For example, the module is a tad-ata-typetoxin-antitoxin module. For example, the module is a plasmid addictionmodule. In an example, the first and/or second cell is a Bacteroidescell and the module is a module of a Bacteroides species, eg, theTxe/YoeB family addiction module (see, eg,http://www.uniprot.org/uniprot/F0R9D1); RelE/StbE family addictionmodule (see, eg, http://www.uniprot.org/uniprot/F0R9A0); HigA familyaddiction module (see, eg, http://www.uniprot.org/uniprot/D7J8V2 orhttp://www.uniprot.org/uniprot/D2ESD0); RelE/StbE family addictionmodule (see, eg, http://www.uniprot.org/uniprot/F0R5F4). Use of atoxin-antitoxin in the vector or MGE can be useful to allow fordestruction of a vector-bearing cell other than a cell that is desired(eg, the first and second and/or third bacterial cell). In this example,the MGE or vector comprises a toxin gene of a bacterial toxin-antitoxinmodule and a cognate anti-toxin gene, wherein the expression of thetoxin and anti-toxin genes are separately regulated, eg, from differentpromoters. For example, the toxin gene can comprise a promoter that isconstitutively active in the first, second (and third) cells so that thetoxin is always produced. The anti-toxin gene can comprise a promoterthat is inducible by one or more factors (eg, a protein expressed) inthe first and/or second cells, but not in non-target cells of differentstrain or species. As is known, the anti-toxin is inherently less stablethan the toxin in a bacterial toxin/anti-toxin system, and thus transferof the vector or MGE to a cell that is not a target cell (eg, not thefirst and/or second cell) will lead to toxin expression in the absenceof anti-toxin expression or lower anti-toxin activity, thus leading tocell death of the non-target cell. This, therefore creates a selectionpressure for the target cells (first, second and third cells) to take upand retain the vector of the invention so that it can have the desiredCRISPR array activity therein and also be propagated across target cellsin a population (such as the gut microbiota). This also limits thespread of the vector or MGE to non-target cells so that the effect ofthe array is controlled in the population—in this respect there will bea pressure for non-target cells not to take up the vector and if theydo, the recipient cells will not survive in the population, therebylimiting replication of non-target cells with the MGE and array.

31. The vector of any preceding embodiment wherein the first and secondcells are of the same phylum (eg, both bacterial cells) and the vectoris replicable or operable (d) in the first cell and/or second cell butnot in another cell of the same phylum; (e) in the first cell and/orsecond cell but not in another cell of the same order; (f) in the firstcell and/or second cell but not in another cell of the same class; (g)in the first cell and/or second cell but not in another cell of the sameorder; (h) in the first cell and/or second cell but not in another cellof the same family; (i) in the first cell and/or second cell but not inanother cell of the same genus; (j) in the first cell and/or second cellbut not in another cell of the same species; (k) in the first celland/or second cell but not in another cell of the same strain. Thisaffords selectivity of the vector of the invention (eg, for selectivekilling of the second host cell type in a mixed bacterial population) ina microbiome. This can be achieved, for example, by engineering the MGEor array (eg, the promoter thereof) so that it requires expression of aparticular protein for replication or operation (eg, expression toproduce crRNA). For example, the promoter can be selected from apromoter that operates in the first and/or second cell but not in othercells, or wherein the MGE is engineered so that one or more of thereplication initiation sites thereof are dependent upon a protein orother factor produced in the first and/or second cell but in not othercells.

32. First and second copies of the vector of any preceding embodiment ina mixed population of cells, wherein the first vector is comprised bythe first cell, the second vector is comprised by the second cell, thecells are cells of different species (eg, different bacterial species)and the one or both of the vector MGEs is capable of transferring to athird cell (eg, a bacterial cell), wherein the third cell species is thesame as the species of the first or second cell or is a species that isdifferent from the first and second cell species. This is useful, sincethe first cell can act as a carrier (eg, when it is non-pathogenic itcan be adminstered to a human or animal so that it populates the humanor animal, such as a microbiome thereof). By horizontal transfer, thecarrier can transfer and propagate CRISPR arrays of the invention tothird cells (directly or via second cells, the latter acting as areservoir for arrays). The arrays can then mediate Cas modification (eg,cutting) of the target sequence in the third cells, eg, to inactivate ordown-regulate an essential or antibiotic resistance gene of the thirdcells.

Generally herein, when the target sequence is comprised by an antibioticresistance gene of a cell, the vector, engineered sequence or array ofthe invention can be administered to a human or animal together with(simultaneously or sequentially) the antibiotic. This is useful to killor reduce proliferation of cells comprising the target sequence. In thisrespect, the vector, engineered sequence or array is comprised by acomposition comprising an antibiotic, wherein the target sequence is asequence of a gene encoding for resistance to said antibiotic.

Optionally, the mixed population comprises the third cell.

In an example, there is a provided a plurality of the first cells, eachcomprising a vector of the invention. In an example, there is a provideda plurality of the second cells, each comprising a vector of theinvention. In an example, there is a provided a plurality of the firstcells in combination with a plurality of the second cells, each cellcomprising a vector of the invention. In an example, there is a provideda plurality of the first cells in combination with a plurality of thesecond cells and a plurality of the third cells, cells of at least 2 (orall of) said pluralities comprising a vector of the invention.

33. The vectors of embodiment 32, wherein the vector or MGE comprises atoxin-antioxin module that is operable in the first, second and thirdhost cells; optionally wherein the toxin-antitoxin module comprises ananti-toxin gene that is not operable or has reduced (ie, lesser)operation in cells other than the first, second and third cells.

34. The vector of any preceding embodiment, wherein the MGE is aconjugative transposon, oriT is functional in the first and second hostcells, the MGE comprises first and second terminal repeat sequences andthe CRISPR array between the repeat sequences, and wherein the first andsecond cells are bacterial cells, the second cell being of a humanmicrobiota cell species (eg, a pathogenic species), wherein the targetsite is comprised by the second cell but not the first cell, and whereinsaid modifying inactivates or down-regulates a gene or regulatorysequence comprising said target in the second cell.

Usefully, the first cells can thereby act as carriers and reservoirs forthe arrays of the invention, which can be transferred by horizontaltransfer of the MGEs.

In an example, the MGE is a conjugative Bacteroidetes transposon, oriTis a Bacteroidetes oriT functional in the first and second host cells,the MGE comprises first and second terminal repeat sequences and theCRISPR array between the repeat sequences, and wherein the first andsecond cells are bacterial cells, the first cell being a Bacteroidetescell and the second cell being a Firmicutes cell (eg, Clostridium orStaphylococcus cell), wherein the target site is comprised by the secondcell but not the first cell, and wherein said modifying inactivates ordown-regulates a gene or regulatory sequence comprising said target inthe second cell.

35. The vector of embodiment 34 when comprised by the first or secondcell.

36. The vector of any preceding embodiment, wherein the first and secondcells are comprised by a mixed bacterial cell population, eg, apopulation of cells of human or non-human animal (eg, dog, cat or horse)gut, vaginal, armpit or oral microbiota species. As explained above, thepopulation is useful for administration to a human or animal to populatea microbiome thereof.

37. An ex vivo composition comprising a plurality of cells as defined inembodiment 22, wherein each cell comprises a vector according to any oneof embodiments 1 to 36. Alternatively, the composition is in vivo, eg,in a non-human animal.

38. A beverage or foodstuff for human or non-human animal consumptioncomprising a vector of any one of embodiments 1 to 36 or the compositionof embodiment 37. The beverage can be, for example, a probiotic drink,eg, for consumption daily, once every two days or weekly by a human oranimal, eg, to treat or prevent obesity or a GI condition in the humanor animal.

39. A composition comprising a plurality of Bacteroides cells, whereineach cell comprises a vector according to any one of embodiments 1 to36.

Usefully, the cells can act as carriers and a reservoir of arrays of theinvention, for administration to a microbiome (eg, gut microbiome) of ahuman or animal, eg, to treat or prevent obesity or a GI condition inthe human or animal,

40. A mixed population of bacterial cells comprising a sub-population offirst cells and a sub-population of second cells, wherein the firstcells comprise vectors according to any one of embodiments 1 to 36,wherein the vectors are capable of horizontal transfer between the firstand second cell sub-populations. Such a population is useful as it canbe adminstered (eg, intranasally) to a human or animal so that thebacteria populate one or more microbiomes (eg, gut microbiome) of thehuman or animal. The first (and optionally also the second) cells canact as carriers of the CRISPR arrays of the invention, especially whenthose cells are non-pathogenic to the human or animal (eg,non-pathogenic in the gut microbiome). The microbiome can be any othermicribiome or microbiota population disclosed herein.

41. The population of embodiment 40, wherein one or both of the firstand second bacterial species is capable of populating the gut microbiotaof a human or non-human animal, and optionally the first bacteria arecommensal or symbiotic with humans or animals. Usefully, the firstbacteria can be safely administered to the human or animal and can actas a carrier of the arrays of the invention for transfer thereafter toother cells of the microbiota.

42. The population of embodiment 40, wherein the mixed population isharboured by a beverage or water (eg, a waterway or drinking water forhuman consumption) or soil. Provision of the population in water or soilis useful for treating such in the environment or (for water) inheating, cooling or industrial systems, or in drinking water storagecontainers.

In an example of any embodiment, the second cell is a cholera cellcomprising the target sequence, wherein when the target sequence ismodified the cell is killed or cell proliferation is reduced. In anexample, the second cell is comprised by water for human consumption(eg, such water before or after processing for human consumption). In anexample, the vector is comprised by a pharmaceutical composition foradministration to a human to treat or prevent cholera in the human.

43. A composition comprising a plurality of vectors according to any oneof embodiments 1 to 36 in vitro. For example, the composition is mixedwith a multi-species bacterial population in an industrial apparatus orcontainer (eg, for food, consumer goods, cosmetics, personal healthcareproduct, petroleum or oil production).

44. The vector, composition, foodstuff, beverage or population of anypreceding embodiment for administration to a human or non-human animalfor therapeutically or prophylactically populating and rebalancing amicrobiome thereof or for cosmetically changing the human or animal (eg,for cosmetic weight-loss).

45. A method of modifying a target nucleotide sequence in a host cell,the method comprising

(1) combining the host cell with a carrier cell,(a) wherein the carrier cell comprises a CRISPR nucleic acid vectorcomprising a CRISPR array for modifying the target,(b) wherein the CRISPR array comprises one or more sequences forexpression of a crRNA and a promoter for transcription of thesequence(s) in the host cell;(c) wherein the crRNA is capable of hybridising to the target sequenceto guide Cas (eg, a Cas nuclease) in the host cell to modify the targetsequence; and(2) culturing the cells together, wherein the vector is transferred fromthe carrier cell to the host cell, whereby the crRNA hybridises to thetarget sequence to guide Cas in the host cell and the target ismodified.

In an example, the method is carried out ex vivo. In an example, themethod is a cosmetic method and is not a therapeutic or prophylacticmedical method.

46. The method of embodiment 45, wherein the vector is according to anyone of embodiments 1 to 36.

47. The method of embodiment 45 or 46, wherein the host cell is a cellof a human or non-human animal microbiome bacterial species, optionallywherein the host cell is a cell of a pathogenic bacterial species. In anexample, any microbiome herein is selected from a gut, vaginal, armpit,scalp, skin or oral microbiome.

48. The method of any one of embodiments 45 to 47, wherein the carriercell is of a species that is a commensal or symbiotic human or non-humananimal microbiome bacterial species. In an example, the carrier cell isnon-pathogenic to humans, eg, when administered intranasally, topicallyor orally.

In any configuration, concept, aspect, embodiment or example etc hereinthe vector, composition, array or population of the invention isadministered intranasally, topically or orally to a human or non-humananimal, or is for such administration. The skilled person aiming totreat a microbiome of the human or animal will be able to determine thebest route of administration, depending upon the microbiome of interest.For example, when the microbiome is a gut microbiome, administration canbe intranasally or orally. When the microbiome is a scalp or armpitmicrobiome, administration can be topically. When the microbiome is inthe mouth or throat, the administration can be orally.

49. The method of any one of embodiments 45 to 48, wherein the host cellis of a gut microbiome bacterial species of a human or non-human animal.

50. A method of altering the relative ratio of sub-populations of firstand second bacteria host cell species in a mixed population of bacteriacomprising said sub-populations, the method comprising

A: providing said first bacterial host cells;B: providing the second bacterial host cells, wherein the second cellsare cells of a different species or strain to the first cells;C: introducing engineered CRISPR arrays into the first bacterial hostcells, wherein each CRISPR array comprises one or more sequences forexpression of a crRNA and a promoter for transcription of thesequence(s) in a said second host cell, wherein the crRNA is capable ofhybridising to a target sequence comprised by said second cell to guideCas (eg, a Cas nuclease) in the host cell to modify the target sequence;D: combining the first and second bacterial cells together to produce amixed bacterial population; andE: allowing bacterial growth in the mixed population such thathorizontal transfer of CRISPR arrays from first bacterial cells tosecond bacterial cells occurs, wherein target sequences in second cellsare Cas modified, whereby the relative ratios of said first and secondbacteria is altered.

51. The method of embodiment 50, wherein each CRISPR array is accordingto any one of embodiments 1 to 26.

52. The method of embodiment 50 or 51, further comprising obtaining afirst sample of the mixed population of step E and optionally comparingthe proportion of second cells in the first sample to the proportion ofsecond cells in a second sample of cells, wherein the second sample is asample of a mixed population of bacterial cells used to provide thesecond cells in step B and the comparison shows that the proportion ofsecond cells has increased or decreased after step E.

53. The method of embodiment 52, wherein the second sample is a sampleof a human or animal microbiome (eg, gut, vaginal, scalp, armpit, skinor oral cavity cells).

54. The method of any one of embodiments 50 to 53, wherein a sample of ahuman or animal microbiome (eg, gut, vaginal, scalp, armpit, skin ororal cavity cells) is used to provide the second cells of step B.

55. The method of any one of embodiments 50 to 54, wherein arecombinant, cultured population of the first cells is used for step A.

56. The method of any one of embodiments 50 to 55, wherein plasmid, ICEor transposon horizontal transfer is used in step E, wherein eachplasmid, ICE or transposon comprises a said CRISPR array.

57. The method of any one of embodiments 50 to 56 for therapeutically orprophylactically rebalancing the microbiota of a human or non-humananimal, eg, for treating or preventing obesity, diabetes IBD, a GI tractcondition or an oral cavity condition. The diabetes can be Type I or II.In an example, the prophylaxis is medical. In an example, theprophylaxis herein is non-medical, eg, cosmetic or for hygiene purposes.For example, the microbiota is an armpit microbiota and the method isfor preventing or reducing body odour of a human. For example, in thiscase the method down-regulates growth or viability of host bacterialcells that mediate the generation and/or persistence of human bodyodour.

58. The method of any one of embodiments 50 to 57, comprising providingthird bacterial host cells of a species or strain that is different tothe carrier and host cells, wherein the third cells are comprised by themixed population in step E or combined with said population after stepE, wherein horizontal transfer of CRISPR arrays to third host cellsoccurs.

59. The method of embodiment 58, wherein the third cells do not comprisea said target sequence. In this way, the third cells can act as carriersof the arrays and are capable of horizontally transferring arrays tohost cells comprising the target sequence.

60. The method of embodiment 58, wherein the third cells do comprise atarget sequence for Cas modification.

61. The method of any one of embodiments 50 to 60, wherein the carrier(and optionally also the third) cells are of a species recited inembodiment 21, eg, Bacteroidetes cells.

62. The method of any one of embodiments 50 to 60, wherein the hostcells are of a species recited in embodiment 19 or Firmicutes cells.

63. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment wherein each vector is or is comprised by aplasmid, phage (eg, a packaged phage) or phagemid.

64. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment, wherein the modifying is (i) cutting of thetarget sequence, (ii) down-regulating transcription of a gene comprisingthe target sequence, (iii) up-regulating transcription of a genecomprising the target sequence, or (iv) adding, deleting or substitutinga nucleic acid sequence at the target.

65. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment, wherein each target sequence is a sequencecomprised by a regulatory element or gene of the host cell, wherein thegene is an essential gene, a CRISPR gene or an antibiotic resistancegene, optionally wherein the regulatory element is an element of such agene. In an alternative, the gene is a virulence gene.

66. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment, wherein each target sequence is a sequencecomprised by a phage genome, wherein the phage is comprised by the hostcell. In an example, the target sequence is comprised by a phage generequired for host cell infectivity, the phage lysogenic or lytic cycle,or phage viability, eg, an essential gene or coat protein gene.

In an example, the Bacteroidetes phage is a Bacteroides phage selectedfrom a crAssphage, a GB-124 phage, a GA-17 phage, a HB-13 phage, aH16-10 phage, a B40-8 phage and B fragalis phage ATCC51477-B1. This isuseful, for example, for providing a survival advantage to Bacteroidetesin the gut microbiome of a human or animal. In this way, the ratio ofBacteroidetes to Firmicutes can be altered to increase the proportion ofthe former versus the latter (eg, for treating or preventing obesity).In an example, the target sequence is comprised by a BACON(Bacteroidetes-associated carbohydrate-binding) domain-encoding sequence(eg, wherein the host is a Bacteroides host) or an endolysin-encodingsequence.

67. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment, wherein each CRISPR array comprises asequence R1-S1-R1′ for expression and production of the respective crRNAin the host cell,

(i) wherein R1 is a first CRISPR repeat, R1′ is a second CRISPR repeat,and R1 or R1′ is optional; and(ii) S1 is a first CRISPR spacer that comprises or consists of anucleotide sequence that is 95% or more identical to said targetsequence.

68. The vector, composition, foodstuff, beverage, population or methodof embodiment 67, wherein R1 and R1′ are at least 95% identicalrespectively to the first and second repeat sequences of a CRISPR arrayof the second host cell species.

69. The vector, composition, foodstuff, beverage, population or methodof embodiment 67 or 68, wherein R1 and R1′ are functional with aCRISPR/Cas system of said host cell for modification of the targetsequence.

70. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment, wherein the or each array is in combinationwith one or more Cas nuclease(s) that function with the respective crRNAin a host cell to modify the target sequence. The target sequencecomprises a protospacer sequence immediately adjacent to a ProtospacerAdjacent Motif (PAM).

71. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment, wherein the or each array is in combinationwith nucleic acid sequence(s) encoding one or more Cas nuclease(s) thatfunction with the respective crRNA in a host cell to modify the targetsequence.

72. The vector, composition, foodstuff, beverage, population or methodof any one of embodiments 67 to 71, wherein R1 and R1′ are functionalwith a Type II Cas9 nuclease (eg, a S pyogenes or S aureus Cas9) tomodify the target in a said host cell, optionally wherein the vector,composition, foodstuff, beverage, population or method is furtheraccording to embodiment 70 or 71 wherein the Cas is said Cas9.

73. An ex-vivo mixed population of bacteria obtainable by the method ofany one of embodiments 50 to 72.

74. A composition for administration to a human or non-human animal fortherapeutic, prophylactic, cosmetic, human or non-human animal body massreduction (eg, cosmetic reduction) or nutritional use, the compositioncomprising the mixed population of embodiment 73.

75. A foodstuff or beverage for human or non-human animal consumptioncomprising the mixed population of embodiment 73 or the composition ofembodiment 74.

76. The foodstuff or beverage of embodiment 75, which is a nutritionalsupplement or a probiotic beverage or foodstuff.

77. An antibiotic composition for treating or preventing a bacterialinfection in a human or non-human animal or in drinking water or insoil, wherein the composition comprises a vector of any one ofembodiments 1 to 36 and 63 to 72.

78. A probiotic composition for increasing the proportion of gutBacteroidetes (eg, to treat or prevent obesity, diabetes or a GIinflammatory condition) in a human or non-human animal, wherein thecomposition comprises a vector of any one of embodiments 1 to 36 and 63to 72.

79. The composition of embodiment 74, 77 or 78 for increasing therelative proportions of gut Bacteroides to Fermicutes in a human oranimal, eg for treating or preventing obesity, diabetes or a GIcondition.

80. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment, wherein the vector does not comprise a Casnuclease-encoding sequence operable with the array. This is useful tosave space in the vector (eg, to allow for inclusion of larger arrays ormore arrays for host cell targeting—this is useful to target multiplegenome locations to reduce likelihood of evolution of resistance to thearrays of the invention).

81. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment, wherein the MGE does not comprise a Casnuclease-encoding sequence operable with the array. This is useful tosave space in the MGE (eg, to allow for inclusion of larger arrays ormore arrays for host cell targeting—this is useful to target multiplegenome locations to reduce likelihood of evolution of resistance to thearrays of the invention). For example, it is possible to avoid includingthe large sequence encoding Cas9 endounclease.

82. The vector, composition, foodstuff, beverage, population or methodof embodiment 80 or 81, wherein the array is operable with a Casendonuclease found in cells of the same species or strain as the firstand/or second cell. In an example, the array is operable with a Casendonuclease found in cells of the same species or strain as a host cellor third cell. This is useful to save space in the vector or MGE (eg, toallow for inclusion of larger arrays or more arrays for host celltargeting—this is useful to target multiple genome locations to reducelikelihood of evolution of resistance to the arrays of the invention).

83. The vector, composition, foodstuff, beverage or population of anypreceding embodiment, wherein the first and second cells are bacterialcells of different species, wherein the second cell is of a humanmicrobiota species and the first cell is of a species that isnon-pathogenic in said human microbiota, wherein the target sequence isnot comprised by the genome of the first cell, the MGE comprising anoriT that is operable in the first and second cells, wherein the MGE iscapable of horizontal transfer from the first cell to the second cell.

In an alternative, there is provided:—

The method of any preceding embodiment, wherein the carrier and hostcells are bacterial cells of different species, wherein the host cell isof a human microbiota species and the carrier cell is of a species thatis non-pathogenic in said human microbiota, wherein the target sequenceis not comprised by the genome of the carrier cell, the MGE comprisingan oriT that is operable in the carrier and host cells, wherein the MGEis capable of horizontal transfer from the carrier cell to the hostcell.

84. The vector, composition, foodstuff, beverage, population or methodof claim 83, wherein the vector is comprised by a bacteriophage, thebacteriophage being capable of infecting the first cell (carrier) tointroduce the MGE into the first (carrier) cell.

85. The vector, composition, foodstuff, beverage, population or methodof embodiment 83 or 84, wherein the target sequence is comprised by thegenome of the second (host) cell (eg comprised by an essential orantibiotic resistance gene of the genome).

86. The vector, composition, foodstuff, beverage, population or methodof embodiment 85, wherein the second (host) cell species is pathogenicin said human microbiota, wherein the target sequence is modified bycutting of the target sequence or down-regulating a gene comprising saidtarget sequence. In an example, the second (host) cell is a cellaccording to any one of features (i) to (xiv) of embodiment 19. In anexample the second (host) cell is a Firmicutes cell, eg, wherein thevector is for treating or preventing obesity in a human.

87. The vector, composition, foodstuff, beverage, population or methodof embodiment 83, 84 or 85, wherein the second (host) cell species isnon-pathogenic in said human microbiota.

88. The vector, composition, foodstuff, beverage, population or methodof any one of embodiment 83 to 87, wherein the second (host) cell is aBacteroidetes or Prevotella cell; optionally wherein the MGE is capableof horizontal transfer from the second (host) cell species to Firmicutesspecies of said human microbiota. The latter is useful, for example, fortreating or preventing obesity in a human when the target sequence iscomprised by the Firmicutes, but not the first (carrier) or second(host) cell.

89. The vector, composition, foodstuff, beverage, population or methodof any one of embodiment 83 to 88, wherein the MGE is capable ofhorizontal transfer from the second (host) cell species to a thirdbacterial cell species of said human microbiota, wherein the third cellspecies is pathogenic in said human microbiota and comprises said targetsequence. In an example, the first (carrier) and second (host) cells donot comprise the target sequence.

90. The vector, composition, foodstuff, beverage, population or methodof embodiment 89, wherein the third cell is a cell according to any oneof features (i) to (xiv) of claim 19.

91. The vector, composition, foodstuff, beverage, population or methodof any preceding embodiment, wherein the MGE is devoid of a sequenceencoding a Cas endonuclease that is operable with repeat sequences ofthe array, and wherein the vector comprises such a sequence (eg,encoding a Cas9) outside the MGE.

Any of the general features also may apply to the present configuration.Any of the features of any other configuration, aspect, paragraph,example, embodiment or concept herein also may be combined with thepresent configurations employing MGEs.

Thus, the invention provides the following features, numbered asparagraphs; these paragraphs apply to any of the aspects as recited, orto any of embodiments 1 to 91, or to any other configuration herein:—

1. A vector of any one of aspects 44 to 50, wherein the target sequenceis a nucleotide sequence of a host CRISPR/Cas system, whereby the crRNAguides Cas to the target to modify the host CRISPR/Cas system in thehost cell.

2. The vector of paragraph 1, wherein the host CRISPR/Cas system is aType I, II or III system and the target sequence is a nucleotidesequence conserved in said Type of system in at least one, two or threeadditional host strains or species, wherein said additional strains orspecies are different from said host.

3. The vector of any preceding paragraph, wherein the target sequence isidentical to a Streptococcus species (eg, S thermophilus or S pyogenes)CRISPR/Cas system sequence.

4. The vector of any preceding paragraph, wherein the target sequence ofthe host CRISPR/Cas system comprises

i. a CRISPR array leader or leader promoter sequence contiguous with the5′-most nucleotide of the first repeat (and optionally comprising said5′-most nucleotide of the repeat, eg, comprising the first 3 nucleotidesat the 5′ end of the first repeat);

ii. a sequence of up to 20 (eg, 3, 5, 7, 9, 10, 12, 15, 20, 30 or 32)nucleotides contiguous nucleotides immediately 5′ of the first repeat;

iii. a sequence of up to 20 (eg, 3, 5, 7, 9, 10, 12, 15, 20, 30 or 32)contiguous nucleotides of the 5′-most nucleotides of the first repeat;or

iv. a sequence of up to 20 (eg, 3, 5, 7, 9, 10, 12, 15, 20, 30 or 32)contiguous nucleotides immediately 3′ of the first spacer repeat (andoptionally wherein the sequence comprises the 3′-most nucleotide of thefirst spacer, eg, comprising the last 3 nucleotides at the 3′ end of thefirst repeat).

5. The vector of paragraph 1, 2 or 3, wherein the array is comprised bya nucleic acid vector (eg, a virus, virion, phage, phagemid or prophage)and

i. the crRNA comprises or consists of the structure R—S—R, wherein R=aCRISPR repeat and S=a CRISPR spacer, wherein S comprises, (in 5′ to 3′direction) V-H_(R) or H_(R)-V or, wherein V=a sequence identical to aDNA sequence of the vector and H_(R)=a DNA sequence of a repeat of aCRISPR array of said host cell CRISPR array;

ii. wherein the sequence of H_(R) is immediately contiguous with thesequence of V in the host CRISPR array; and

iii. wherein the crRNA is capable of hybridising to a spacer of the hostCRISPR array to guide Cas to the host target for modification of thehost CRISPR array in the cell.

For example, V is a sequence of a phage vector coat protein-encodingsequence. In this respect Heler et al found in a study of bacterialresistance that three CRISPR-independent, bacteriophage-resistantmutants displayed a marked defect in phage adsorption (about 50%),indicating that most likely they carry envelope resistance mutations.

6. The vector of paragraph 5, wherein the first crRNA does not or doesnot substantially hybridise to the nucleic acid present in the vector.For example, the first crRNA does not hybridise to V in the vector orhybridises less strongly than it hybridises to the spacer of the hostarray. Hybridisation testing is routine for the skilled person. Forexample, it can be determined in vitro by isolating or synthesizing thevector DNA and incubating it with the crRNA. Standard techniques, eg,using PCR can be used to detect whether or not hybridisation hasoccurred (eg, tested under pH and temperature conditions that would befound in host cell).

7. The vector of paragraph 5 or 6, wherein V=one or up to 40 (eg, up to15) contiguous nucleotides of vector DNA. The seed sequence immediately5′ of the PAM in the protospacer found in a target sequence is importantfor crRNA pairing and functioning of the CRISPR/Cas system to cut. Thisseed sequence includes around 15 or 12 contiguous nucleotidesimmediately 5′ of the PAM.

8. The method, array or vector of any preceding aspect or paragraph,wherein the array is comprised by a vector and comprises (in 5′ to 3′direction) a first repeat sequence, a first spacer sequence and a secondrepeat sequence, wherein the spacer sequence comprises a sequence thatis capable of hybridising (eg, is identical to or has greater than 90%identity) to the target sequence in the host cell, the array furthercomprising a promoter for transcription of the repeats and spacer in thehost cell, and optionally the vector comprises a Cas nuclease-encodingsequence and/or a tracrRNA-encoding sequence for encoding a functionalCas and/or tracrRNA sequence in the host cell, wherein the tracrRNAsequence comprises a sequence that is complementary to the first orsecond repeat.

9. The method, array or vector of any preceding aspect or paragraph,wherein the CRISPR array is comprised by a vector and comprises (in 5′to 3′ direction) a first repeat sequence, a first spacer sequence and asecond repeat sequence, wherein the spacer sequence comprises a sequencethat is capable of hybridising (eg, is identical to or has greater than90% identity) to the target sequence in the host cell, the array furthercomprising a promoter for transcription of the repeats and spacer in thehost cell, and wherein the vector does not comprise a Casnuclease-encoding sequence and/or a tracrRNA-encoding sequence forencoding a tracrRNA sequence in the host cell wherein the tracrRNAsequence comprises a sequence that is complementary to the first orsecond repeat, wherein the HM-CRISPR array is functional in the hostcell to guide Cas (eg, endogenous host Cas nuclease) to the host targetsite, optionally using a host tracrRNA.

10. The method, array or vector of paragraph 8 or 9, wherein the repeatsare identical to repeats in a host array, wherein the CRISPR array ofthe invention does not comprise a PAM recognised by a Cas (eg, a Casnuclease, eg, Cas9) of a host CRISPR/Cas system. The ability to omit Cassequences frees up space in the array of the invention.

An “essential gene” is a gene in the host whose presence or expressionis required for host cell growth or for promoting or sustaining cellviability. A resistance gene is a gene in the host whose presence orexpression is required for providing complete or partial resistance toan anti-host drug, eg, an antibiotic, eg, a beta-lactam antibiotic. Avirulence gene is a gene in the host whose presence or expression isrequired for infectivity of an organism that the host cell is capable ofinfecting, eg, wherein the host is a pathogen (eg, of a plant, animal,human, livestock, companion pet, plant, bird, fish or insect).

11. The method, array or vector of any preceding aspect or paragraph,wherein the CRISPR array is in combination with a non-host cell Cas (eg,a Type I system Cas wherein the host system is a Type II or III; a TypeII system Cas wherein the host system is a Type I or III; or a Type IIIsystem Cas wherein the host system is a Type I or II), optionallywherein the host cell does not comprise or express a Cas of a Type thatis the same as the Type of the non-host Cas. This is useful since theCRISPR array does not target a sequence in itself (such as in thevector) or a vector-encoded Cas in the host.

12. The method, array or vector of any preceding aspect or paragraph,wherein the CRISPR array is in combination with a tracrRNA sequence or asequence encoding a tracrRNA sequence (eg, on same nucleic acid as thearray), optionally wherein the tracrRNA sequence and HM-crRNA arecomprised by a single guide RNA (gRNA)).

13. The method, array or vector of any preceding aspect or paragraph,wherein the CRISPR array is in combination with a Cas or a sequenceencoding a Cas, optionally wherein the array is integrated in a hostcell genome and the Cas is endogenous to the host cell or encoded by anexogenous sequence. In an example, the Cas-encoding sequence is anexogenous sequence that has been introduced into the host, eg, from aplasmid or virus, such as a phage.

14. The method, array or vector of any preceding aspect or paragraph,wherein the CRISPR array is comprised by a nucleotide sequence of aplasmid, virus, virion, phage, phagemid or prophage. The phagemid is apackaged phage. The prophage is a phage integrated into the hostchromosome or episomal in the cell.

15. The method, array or vector of any preceding aspect or paragraph,wherein the CRISPR array is integrated in a host cell genome, eg, in achromosome or episomal nucleic acid. In one example the array is incombination with a dead Cas (eg, dCas9) conjugated to a transcription ortranslation activator that acts on the target sequence or a genecomprising the target sequence. This is useful, for example, forswitching on gene expression in the host cell (eg, of a desired gene,eg, an exogenous gene sequence that has previously been engineered intothe host cell, eg, to encode an antibiotic where the host is a microbe,or to encode a desired exogenous protein for production in host culture,eg, for food, drink, medicine or any other application of the inventionas disclosed herein).

16. A virus (eg, a virion, phage, phagemid or prophage) comprising aCRISPR array of any preceding aspect or paragraph, eg, for infecting acell, eg, a microbe or for use in medicine or dentistry.

17. A population of virions according to paragraph 16, a first and asecond virion thereof comprising different array leaders or promotersand/or for targeting different target sequences in the host cell or indifferent host strains.

18. A collection of CRISPR arrays, each array being according to anypreceding aspect or paragraph, wherein a first array comprises a firstpromoter for crRNA transcription; a second array comprises a secondpromoter for crRNA transcription that is different from the firstpromoter; and wherein each promoter is identical to a host promoter oris a homologue thereof, optionally wherein the first or both promotersis identical to a host Cas (eg, Cas1, 2, 9 or Csn2) promoter or a hostCRISPR array promoter. For example, the first promoter is an endogenousCas nuclease promoter or endogenous Cas1 or Cas2 promoter; or thepromoter of an endogenous gene that is highly or constitutivelyexpressed or is an essential, virulence or resistance gene of the hostcell. By using endogenous promoters, there will be pressure duringevolution of the host to preserve the host promoters, and thus thisdecreases the likelihood of the host CRISPR/Cas defense system targetingone or more promoters of the arrays.

19. A collection of CRISPR arrays of the invention, wherein a firstarray comprises one or more spacers (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50 or more spacers); and the second array comprises more thanone spacer (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or morespacers), wherein said spacers of the second array are identical to theone or more spacers of the first array. This is useful for evading hostresistance by homologous recombination of HM-array spacers, as provingmany of such spacers in the HM-array (or furthermore distributing thespacers across a plurality of arrays) increases the chances that someHM-array spacers will remain in the host cell even if the host cell doesdelete some of the spacers. The defense against deletion is alsoenhanced by using different repeats flanking identical copies of thespacers in different arrays. Thus the invention provides the following:—

20. The collection of paragraph 18 or 19, wherein spacers (or saidspacers) of the first array are flanked by first repeats that areidentical; spacers (or said spacers) of the second array are flanked bysecond repeats that are identical; and wherein the first repeats aredifferent from the second repeats.

21. The collection of paragraph 20, wherein the first repeats areidentical to repeats in a host cell CRISPR/Cas system.

22. The collection of paragraph 20, wherein the first repeats aredifferent from repeats in a host CRISPR/Cas system.

23. The collection of any one of paragraphs 18 to 22, wherein the firstand second arrays are contained in the same host cell or in the samevector (eg, plasmid, virus, virion, phage, phagemid or prophage).

24. The collection of any one of paragraphs 18 to 22, wherein the firstarray is contained in a first vector and the second array is containedin a second vector which does not contain the first array (eg, whereinthe vectors are plasmids or virions (eg, of the same virus type) orpackaged phage (eg, of the same phage type).

In an embodiment, the vectors used in the method of the invention arevectors comprised by an array of any one of paragraphs 18 to 24.

25. A host cell comprising an array, virus, virion, phage, phagemid,prophage, population or collection according to any preceding paragraph.

Any of the general features (see below) also may apply to the presentconfiguration.

An example of the invention provides the following for reducing the riskof host adaptation and resistance to the array:—

The CRISPR array or vector of the invention for modifying a targetnucleotide sequence of a host cell,

a. wherein the host cell comprises a first endogenous promoter (firsthost promoter) for transcription of the target sequence;

b. wherein the CRISPR array comprises a sequence encoding a crRNA and afirst promoter for transcription of the crRNA, the crRNA beingoptionally comprised by a single guide RNA (gRNA) and capable ofhybridising to the host target sequence to guide Cas to the target inthe host cell to modify the target sequence;

c. wherein the sequence of the first promoter is the sequence of asecond endogenous host promoter that is different to the sequence of thefirst host promoter.

In an example, a promoter is used for each vector (eg, phage) CRISPRunit that is a promoter of an essential gene in the host—that way thehost will express the crRNA well (and constitutively if the promoter isfrom a host gene that must always or often be switched on). The hostwill not easily adapt away from that promoter so will not easily gainresistance. Optionally it is possible to use different essentialpromoters for different vector CRISPR units to decrease the chance ofhost adaptation (resistance). One can use the promoter of the virulenceor essential or resistance gene being targeted in the host by the array(or a different array). To gain resistance to the phage the host wouldneed to mutate the endogenous gene promoter and the gene targeting site(which may, for example, be in an coding sequence that is essential forcell growth, viability or anti-host drug (eg, antibiotic) resistance)and thus risk inactivating the gene that way too.

The provision as per the invention of multiple copies of nucleic acidsequences encoding crRNAs, wherein the copies comprise the same spacersequence for targeting a host cell sequence as per the invention isadvantageous for reducing the chances of host removal (eg, by host cellhomologous recombination) of useful targeting spacers from the vector.Multiple targeting spacers can be provided flexibly, on the same ormultiple HM-arrays of the invention to provide alternative ways ofevading resistance.

Thus, the invention provides the following concepts:—

1. A host modifying (HM) CRISPR/Cas system (eg, Type I, II or III) formodifying a target nucleotide sequence of a host cell, the systemcomprising components according to (i) to (iv):—

(i) at least one nucleic acid sequence encoding a Cas nuclease (eg, aCas9);(ii) an engineered host modifying (HM) CRISPR array (eg, an array asdescribed above) comprising a spacer sequence (HM-spacer) and repeatsencoding a HM-crRNA, the HM-crRNA comprising a sequence that is capableof hybridising to a host target sequence to guide Cas to the target inthe host cell to modify the target sequence;(iii) an optional tracrRNA sequence or a DNA sequence for expressing atracrRNA sequence;(iv) wherein said components of the system comprises two, three or moreof copies (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more); ofnucleic acid sequences encoding crRNAs, wherein the copies comprise thesame spacer sequence for targeting a host cell sequence (eg, a hostvirulence, resistance or essential gene sequence or a sequence of a hostCRISPR/Cas system component that mediates vector adaptation).

For example, the system comprises 4 or more; or 5 or more; of saidcopies of nucleic acid sequences encoding crRNAs comprising the samespacer. This is advantageous to increase the expression of desired cRNAsin the host. Additionally, this provides greater chance of avoiding hostresistance as more than one sequence will need to be targeted(especially if there are may copies such as 5, 10, 15, 20, 30, 40, 50 or100 or more). Distribution of the copies over different arrays, eg, thevector comprises these spaced on the same DNA strand, is useful toreduce the chances of recombination between spacers or between flankingrepeats which could then lead to excision of the desired cRNA-encodingsequences. The chances of the host excising all copies is reduced byproviding copies distributed across many vector arrays, it is alsoreduced by including many copies of the desired spacers (eg, many copiesin a first vector array and many copies in a second vector array—it ispossible to include at least 2, 3, 4, 5, 6, 10 or more such arrays, eachcomprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or 100 or morecopies of the desired spacer).

2. The system of concept 1, wherein said components of the systemcomprises 4, 5, 10, 15 or 20 more of said copies of nucleic acidsequences encoding crRNAs comprising the same spacer.

3. The system of concept 1 or 2, wherein the copies are split betweentwo or more nucleic acid vector CRISPR arrays.

4. The system of concept 3, wherein the system comprises first andsecond HM-arrays, wherein first and second vector CRISPR arrays arecontained in the same host cell or in the same vector (eg, a plasmid,virus, virion, phage, phagemid or prophage).

5. The system of concept 3 or 4, wherein the first array is contained ina first vector and the second array is contained in a second vectorwhich does not contain the first array (eg, wherein the vectors areplasmids or virions (eg, of the same virus type) or phagemids (eg, ofthe same phage type).

6. The system of any preceding concept, wherein the repeats areidentical to repeats in a host CRISPR array.

7. The system of any one of concepts 1 to 5, wherein the repeats are notidentical to repeats in a host CRISPR array.

8. A host cell comprising a system, vector, virus, virion, phage,phagemid or prophage according to any preceding concept.

9. An antimicrobial composition (eg, an antibiotic, eg, a medicine,disinfectant or mouthwash), comprising a system, vector, virus, virion,phage, phagemid or prophage according to any one of concepts 1 to 8.

Any of the general features (see below) also may apply to the presentconcepts.

Split CRISPR/Cas9 System

This configuration is advantageous to free up space in target vectors,for example viruses or phage that have restricted capacity for carryingexogenous sequence. By freeing up space, one is able to include moretargeting spacers or arrays, which is useful for evading hostresistance. It is advantageous, for example to harness the endogenousCas endonuclease rather than encode it in the vector—especially forbulky Cas sequences such as sp or saCas9. Additionally, there is notchance of inferior compatibility as may be seen with some exogenous Casfrom non-host sources. The ability to reduce virus, eg, phage genomesize, may also be beneficial for promoting host cell uptake (infectionand/or maintenance of the virus in host cells). In some examples, anadvantage is that invasion of the host by the vector (eg, phage) mayupregulate host CRISPR/Cas activity, including increased expression ofhost Cas nucleases—in an attempt of the host to combat invading nucleicacid. This, however, is also useful to provide endogenous Cas for usewith the arrays, vectors, systems and other aspects of thisconfiguration invention when these comprise one or more repeats that arerecognised by the host Cas. In the case where the invention involves oneor more spacers targeting a host CRISPR array (as per also the firstconfiguration of the invention), this then promotes inactivation of thehost CRISPR array itself, akin to a “suicidal” host cell which then usesits own Cas nuclease to inactivate its own CRISPR systems. Thus, theinvention provides the following features, numbered as examples:—

1. A host modifying (HM) CRISPR/Cas9 system (eg, Type I, II or III) formodifying a target nucleotide sequence of a host cell, the systemcomprising components according to (i) to (iv):—

(i) at least one nucleic acid sequence encoding a Cas nuclease (eg, aCas9);

(ii) an engineered host modifying (HM) CRISPR array (eg, an array of theinvention described above) comprising a spacer sequence (HM-spacer) andrepeats encoding a HM-crRNA, the HM-crRNA comprising a sequence that iscapable of hybridising to a host target sequence to guide said Cas tothe target in the host cell to modify the target sequence;

(iii) an optional tracrRNA sequence or a DNA sequence for expressing atracrRNA sequence;

(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that can transform the hostcell, whereby the HM-crRNA guides Cas to the target to modify the targetsequence in the host cell.

By “split” here it is meant that the vector comprises one or more (butnot all) of the components of the system and the host cell comprises oneor more (but not all) of the components, and the vector comprises one ormore components that are not comprised by the host cell. In anembodiment, the vector and host cell do not share in common any of thecomponents, eg, the host cell comprises component (i) and the vectorcomprises component (ii), and either the vector comprises component(iii) and/or the host cell comprises component (iii). When the vector isinside the host cell (eg, as an integrated or episomal vector, eg, aprophage), it is intended that the vector is the nucleic acid that hasbeen provided by a vector that has transformed the host cell (andcomponents of the system provided by such nucleic acid are not in thatcase be construed as host cell components). This can readily bedetermined by sequencing of nucleic acid (eg, chromosome and episomalnucleic acid) of the transformed host and comparing this against thesequences from a non-transformed host of the same type (eg, from thesame host parental colony or clone, eg, when the host is a microbe, eg,a bacterium or archaeon).

Optionally, the system is a CRISPR/Cas9 system. Optionally, the nucleaseof (a) is a Type I Cas nuclease. Optionally, the nuclease of (a) is aType II Cas nuclease (eg, a Cas9). Optionally, the nuclease of (a) is aType III Cas nuclease.

2. The system of example 1, wherein at least one of the components isendogenous to the host cell.

3. The system of example 1 or 2, wherein component (i) is endogenous tothe host cell.

4. The system of any one of examples 1 to 3, wherein component (iii) isendogenous to the host cell.

5. A host modifying (HM) CRISPR/Cas system (eg, Type I, II or III) formodifying a target nucleotide sequence of a host cell, the systemcomprising components according to (a) to (e):—

a. at least one nucleic acid sequence encoding a Cas nuclease (eg, aCas9);

b. an engineered host modifying (HM) CRISPR array comprising a spacersequence (HM-spacer) and repeats encoding a HM-crRNA, the HM-crRNAcomprising a sequence that is capable of hybridising to a host targetsequence to guide said Cas to the target in the host cell;

c. an optional tracrRNA sequence or a DNA sequence for expressing atracrRNA sequence;

d. wherein said components of the system are split between at least afirst and a second nucleic acid vector, wherein at the first vectorcomprises component (a) but the second vector lacks component (a); and

e. wherein the vectors can co-transform simultaneously or sequentiallythe host cell, whereby the HM-crRNA guides Cas to the target to modifythe target sequence in the host cell.

-   -   The definition of “split” provided above applies mutatis        mutandis to the present example comprising first and second        vectors.

In an embodiment a tracrRNA sequence is not provided by the vectors, butis a tracrRNA sequence of an endogenous host cell CRISPR/Cas system,wherein the tracrRNA is capable of hybridising with the HM-crRNA in thecell for subsequent processing into mature crRNA for guiding Cas to thetarget in the host cell.

6. The system of example 5, wherein the first vector comprises component(a) and the second vector comprises components (b) and (c).

7. The system of example 5 or 6, wherein the first and/or second vectoreach comprises one, two, three or more further engineeredHM-CRISPR-arrays.

8. The system of any one of examples 5 to 7, wherein one of the firstand second vectors is a phagemid and the other vector is a helper phage.

9. The system of any preceding example (eg, example 3 or 6), wherein thecrRNA sequence and tracrRNA sequence are comprised by a single guide RNA(gRNA), eg provided by the vector.

10. The system of any preceding example, wherein each vector has arestricted capacity for insertion of exogenous nucleic acid.

11. The system of any preceding example, wherein the vector or vectorsare viruses (eg, virions, packaged phage, phagemid or prophage).

12. The system of any preceding example, wherein the host cell comprisesa deoxyribonucleic acid strand with a free end (HM-DNA) encoding aHM-sequence of interest and/or wherein the system comprises a sequenceencoding the HM-DNA (eg, integrated in the vector or in the host cellgenome or an episome thereof), wherein the HM-DNA comprises a sequenceor sequences that are homologous respectively to a sequence or sequencesin or flanking the target sequence.

The strand comprises a free end, ie, an end not integrated into the hostor vector DNA such that the strand has one or two free ends, ie, the DNAis unbonded to a neighbouring nucleotide immediately 5′ and or 3′respectively.

13. The system of example 12, wherein the target site is cut in the hostcell by Cas (eg, by Cas9 when said Cas nuclease is a Cas9), and theHM-DNA comprise first and second sequences that are homologous 5′ and 3′respectively flanking the cut for inserting the HM-DNA into the hostgenome (eg, into a chromosomal or episomal site).

14. The system of example 13, wherein the insertion is by homologydirected recombination (HDR).

15. The system of example 13, wherein the insertion is by non-homologousend joining (NHEJ).

16. The system of any one examples 12 to 15, wherein the HM-sequence isor encodes a regulatory element (eg, a promoter, eg, an induciblepromoter that replaces an endogenous promoter), a transcriptioninhibiting sequence, a transcription enhancing sequence, a label, or asequence that encodes an exogenous protein or domain.

17. The system of any one of examples 12 to 16, wherein the systemcomprises first and second HM-DNAs wherein a sequence of the firstHM-DNA is complementary to a sequence of the second DNA whereby the DNAsare able to combine in the host cell by homologous recombination to forma combined HM-DNA for insertion into the host cell genome (eg, into achromosomal or episomal site).

18. The system of any preceding example, wherein the vector or vectorsare capable of infecting the host cell to introduce vector nucleic acidcomprising a system component into the cell.

19. The system of any preceding example, wherein said Cas nuclease is anickase.

20. The system of any preceding example, wherein the cell is a bacteriaor archaea and said Cas nuclease is provided by an endogenous Type IICRISPR/Cas system of the bacteria or archaea.

21. The system of any preceding example, wherein the vector or vectorsare inside a said host cell, optionally integrated into a host DNA.

22. The system of any preceding example, wherein the vector or vectorslack a Cas nuclease (eg, aCas9)-encoding sequence.

23. An engineered nucleic acid viral vector (eg, a vector, virion orpackaged phage as described above) for infecting a microbe host cellcomprising an endogenous CRISPR/Cas system, the vector

(a) comprising nucleic acid sequences for expressing a plurality ofdifferent crRNAs for use in a CRISPR/Cas system according to anypreceding example; and

(b) lacking a nucleic acid sequence encoding a Cas nuclease (eg, aCas9), wherein a first of said crRNAs is capable of hybridising to afirst nucleic acid sequence in said host cell; and a second of saidcrRNAs is capable of hybridising to a second nucleic acid sequence insaid host cell, wherein said second sequence is different from saidfirst sequence; and

(c) the first sequence is comprised by an anti-microbe (eg, antibiotic)resistance gene (or RNA thereof) and the second sequence is comprised byan anti-microbe resistance gene (or RNA thereof); optionally wherein thegenes are different;

(d) the first sequence is comprised by an anti-microbe resistance gene(or RNA thereof) and the second sequence is comprised by an essential orvirulence gene (or RNA thereof);

(e) the first sequence is comprised by an essential gene (or RNAthereof) and the second sequence is comprised by an essential orvirulence gene (or RNA thereof); or

(f) the first sequence is comprised by a virulence gene (or RNA thereof)and the second sequence is comprised by an essential or virulence gene(or RNA thereof).

24. An engineered (directly engineered or isolated from a vector in ahost cell, where that vector was derived from an engineered vector thattransformed the host) nucleic acid vector for transforming a host cellcomprising an endogenous CRISPR/Cas system, the vector optionally beinga vector as described above and

(a′) comprising nucleic acid sequences for expressing a plurality ofdifferent crRNAs for use in a CRISPR/Cas system according to anypreceding example; and(b′) lacking a nucleic acid sequence encoding a Cas nuclease (eg, aCas9), wherein a first of said crRNAs is capable of hybridising to afirst nucleic acid sequence in said host cell; and a second of saidcrRNAs is capable of hybridising to a second nucleic acid sequence insaid host cell, wherein said second sequence is different from saidfirst sequence; and the first and/or second sequence is a targetsequence of the host CRISPR/Cas system which sequence is or comprises(c′) a repeat DNA or RNA sequence (eg, wherein the repeat is the 5′-mostrepeat (the first repeat) in said host CRISPR array;(d′) a tracrRNA sequence or a tracrRNA-encoding DNA sequence;(e′) a CRISPR array leader sequence;(f′) a Cas gene promoter (eg, a Cas1, Cas2 or Csn2 promoter);(g′) a CRISPR array leader promoter sequence; or(h′) a Cas-encoding DNA or RNA sequence (eg, wherein the Cas is Cas9,Cas1, Cas2 or Csn2), eg, wherein a first of said crRNAs is capable oftargeting a host Cas1 gene sequence (or a sequence of an RNA thereof)and a second of said crRNAs is capable of targeting a host Cas2 genesequence (or a sequence of an RNA thereof).

25. The vector of example 24, wherein the first and/or second targetsequence is or comprises

i. a CRISPR array leader or leader promoter sequence contiguous with the5′-most nucleotide of the first repeat (and optionally comprising said5′-most nucleotide of the repeat), eg, comprising the first 3nucleotides at the 5′ end of the first repeat;

ii. a sequence of up to 20 (eg, 3, 5, 7, 9, 10, 12, 15, 20, 30 or 32)contiguous nucleotides immediately 5′ of the first repeat;

iii. a sequence of up to 20 (eg, 3, 5, 7, 9, 10, 12, 15, 20, 30 or 32)contiguous nucleotides of the 5′-most nucleotides of the first repeat;or

iv. a sequence of up to 20 (eg, 3, 5, 7, 9, 10, 12, 15, 20, 30 or 32)contiguous nucleotides immediately 3′ of the first spacer (andoptionally wherein the sequence comprises the 3′-most nucleotide of thefirst spacer), eg, comprising the last 3 nucleotides at the 3′ end ofthe first repeat.

26. The vector of example 24 or 25, wherein the or each target sequenceis comprised by a sequence selected from the group consisting of SEQ IDNO: 1 to 44, or a complement thereof.

27. The vector of any one of examples 24 to 26, wherein the first crRNAcomprises or consists of the structure R—S—R, wherein R=a CRISPR repeatand S=a CRISPR spacer, wherein S comprises, (in 5′ to 3′ direction)V-H_(R) or H_(R)—V or, wherein V=a sequence identical to a DNA sequenceof the vector and H_(R)=a DNA sequence of a repeat of a CRISPR array ofsaid host cell CRISPR/Cas system, wherein the first crRNA is capable ofhybridising to a spacer of the host CRISPR array to guide Cas to thetarget of the crRNA for modification of the host CRISPR array in thecell.

28. The vector of example 27, wherein the first crRNA does notsubstantially hybridise to the nucleic acid present in the vector, eg,wherein the first crRNA does not hybridise to V in the vector orhybridises less strongly than it hybridises to the spacer of the hostarray. The discussion above on determining this applies to this exampletoo.

29. The vector of example 27 or 28, wherein V=one or up to 40 (eg, up to15) contiguous nucleotides of vector DNA. For example, V=1, 2, 3, 4, 5,6, 7 8 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40contiguous nucleotides of vector DNA.

30. The vector of example 29, wherein

i. the host CRISPR/Cas system is able to recognise a cognate PAM;

j. wherein the vector DNA comprises such a PAM immediately 3′ of aprotospacer sequence;

k. wherein V=one or up to 40 (eg, up to 15) nucleotides of theprotospacer; and

l. wherein H_(R)=a sequence identical to a contiguous sequence of therepeat of the host CRISPR array.

31. The vector of example 30, wherein said contiguous sequence of therepeat of the host array is a sequence of at least 50% of a host repeat(eg, including the 5′-most or 3′-most nucleotide of the host repeat).

32. The vector of example 30 or 31, wherein V=from 1 to 40 (eg, up to15) of the 3′-most protospacer contiguous nucleotides; and optionallysaid contiguous sequence of the repeat includes the 5′-most nucleotideof the host repeat.

33. The vector of example 30 or 31, wherein V=from 1 to 40 (eg, up to15) of the 5′-most protospacer contiguous nucleotides; and optionallysaid contiguous sequence of the repeat includes the 3′-most nucleotideof the host repeat.

34. The vector of any one of examples 27 to 33, wherein R=a repeat thatis recognised by the host CRISPR/Cas system. Alternatively, R=a repeatthat is not recognised by the host CRISPR/Cas system. In this case,preferably the vector comprises a nucleotide sequence of a Cas nuclease(and optionally a tracrRNA) that is cognate to R, ie, is capable offunctioning with R in the host cell.

35. A vector according to any one of examples 24 to 34, wherein thefirst sequence is according to any one of (c′) to (h′) and the secondsequence is selected from a host essential gene, virulence gene orresistance gene.

36. An engineered nucleic acid viral vector (eg, a virion or packagedphage) for use in the system of any one of examples 1 to 22 forinfecting a microbe host cell comprising an endogenous CRISPR/Cassystem,

a. the vector comprising a first nucleic acid sequence for expressing afirst crRNA in the host; andb. wherein the first sequence comprises (in 5′ to 3′ direction)R1a-S1-R1b, wherein R1a=a first CRISPR repeat, wherein R1a is optional;R1b=a second CRISPR repeat and S1=a CRISPR spacer complementary to ahost sequence (eg, a host sequence recited in example 23 or 24), whereinR1a and Rib are recognised by a host Cas nuclease (eg, a Type IInuclease, eg, a Cas9);c. wherein the vector lacks (i) a nucleic acid sequence encoding a Casnuclease (eg, a Cas9) that recognises the repeat(s) of (b) and/or (ii) anucleic acid sequence encoding a tracrRNA sequence that is complementaryto a crRNA sequence encoded by the first sequence. For example, thevector is a nucleic acid vector comprised by a phage.

37. The vector of example 36, wherein

d. the vector comprises a second nucleic acid sequence for expressingsecond crRNA in the host, wherein the second crRNA is different from thefirst crRNA;e. wherein the second sequence comprises (in 5′ to 3′ direction)R2a-S2-R2b, wherein R2a=a first CRISPR repeat, wherein R2a is optional;R2b=a second CRISPR repeat and S2=a CRISPR spacer complementary to ahost sequence (eg, a host sequence recited in example 23 or 24), whereinR2a and R2b are recognised by a host Cas nuclease (eg, a Type I or IInuclease, eg, a Cas6).

Thus, for example, the first and second nucleic acid sequences arecomprised by the same packaged phagemid, eg, in the same or differentCRISPR arrays.

38. The vector of example 37, wherein the vector lacks (iii) a nucleicacid sequence encoding a Cas (eg, a Cas6) that recognises the repeat(s)of (e) and/or (iv) a nucleic acid sequence encoding a tracrRNA sequencethat is complementary to a crRNA sequence encoded by the secondsequence.

39. A collection of engineered nucleic acid viral vectors (eg, vectors,virions or packaged phages as described above) for use in the system ofany one of examples 1 to 22 for co-infecting a microbe host cellcomprising an endogenous CRISPR/Cas system, the collection comprising afirst vector and a second vector,

f. wherein the first vector is according to example 36;g. wherein the second vector comprises a second nucleic acid sequencefor expressing second crRNA in the host, wherein the second crRNA isdifferent from the first crRNA;h. wherein the second sequence comprises (in 5′ to 3′ direction)R2a-S2-R2b, wherein R2a=a first CRISPR repeat, wherein R2a is optional;R2b=a second CRISPR repeat and S2=a CRISPR spacer complementary to ahost sequence, wherein R2a and R2b are recognised by a host Cas nuclease(eg, a Type I or II nuclease, eg, a Cas6).

For example, the first vector is comprised by a first packaged phagemidand the second vector is comprised by a second packaged phagemid.

40. The collection of example 39, wherein the second vector comprises(v) a nucleic acid sequence encoding a Cas (eg, a Cas9) that recognisesthe repeat(s) of (b) and/or (vi) a nucleic acid sequence encoding atracrRNA sequence that is complementary to a crRNA sequence encoded bythe first sequence.

For example, in this case the Cas functions are provided by theendogenous host system. This saves vector space (eg, for inclusion ofmore host-targeting HM-array spacers) and simplifies vector and arrayconstruction.

41. The collection of example 39 or 40, wherein the second vector lacks(vii) a nucleic acid sequence encoding a Cas (eg, a Cas6) thatrecognises the repeat(s) of (h) and/or (viii) a nucleic acid sequenceencoding a tracrRNA sequence that is complementary to a crRNA sequenceencoded by the second sequence. For example, in this case the Casfunctions are provided by the endogenous host system.

42. The collection of example 39, wherein the first and second vectorseach lacks (ix) a nucleic acid sequence encoding a Cas (eg, a Cas9) thatrecognises the repeat(s) of (b) and (x) a nucleic acid sequence encodinga Cas (eg, a Cas6) that recognises the repeat(s) of (h); optionallywherein the collection is comprised by a host cell comprising one ormore Cas that recognise the repeat(s) of (b) and (h).

43. The collection of example 42, further comprising a third vector (eg,a virion or a phage) comprising a nucleic acid sequence according to(ix) and/or (x).

44. The collection of any one of examples 39 to 43, wherein each vectoris comprised by a respective packaged virion or phagemid, or arespective virion or phage nucleic acid.

45. The vector or collection of any one of examples 36 to 44, whereinR1a and R1b comprise the same repeat sequence.

46. The vector or collection of any one of examples 37 to 45, whereinR2a and R2b comprise the same repeat sequence.

47. The vector or collection of any one of examples 37 to 46, whereinthe repeat(s) of (b) are recognised by a Cas nuclease that is differentfrom the Cas nuclease that recognises the repeat(s) of (e).

48. The vector or collection of any one of examples 37 to 47, whereinthe host comprises CRISPR/Cas systems of different types (eg, a Type Iand a Type II system; a Type I and a Type III system; a Type II and aType III system; or Type I, II and III systems).

49. The vector or collection of any one of examples 36 to 48, whereinthe repeat(s) of (b) are recognised by a Type II Cas nuclease, eg, aCas9.

50. The vector or collection of any one of examples 37 to 49, whereinthe repeat(s) of (e) are recognised by a Type I or III Cas nuclease, eg,a Cas6.

51. The vector or collection of any one of examples 23 to 50, whereinthe vector is a virus, a virion, phage, phagemid or prophage.

52. The vector or collection of any one of examples 23 to 51 inside ahost cell comprising one or more Cas that are operable with cRNA encodedby the vector(s).

53. The vector or collection of any one of examples 23 to 52 inside ahost cell comprising a Cas9.

54. The vector or collection of any one of examples 23 to 53, incombination with a HM-DNA (eg, integrated in the vector, on a plasmid orin the host cell genome or an episome thereof), wherein the HM-DNA is asrecited in any of examples 12 to 17.

55. The system, vector or collection of any preceding example,comprising nucleic acid sequences for expressing a plurality ofdifferent crRNAs, wherein said crRNAs are capable of targeting at least3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or 100 DNA sequences in thehost cell.

56. The system, vector or collection of any preceding example,comprising a first crRNA or a nucleic acid sequence encoding a firstcRNA that is capable of targeting a DNA sequence of a Cas nuclease (orsequence of an RNA thereof) which is not said Cas nuclease (eg, Cas9)but which mediates host vector adaptation; optionally comprising asecond crRNA or a nucleic acid sequence encoding a second cRNA that iscapable of targeting a sequence of a resistance, virulence or essentialhost gene (or RNA thereof) in the host.

57. The system, vector or collection of any preceding example,comprising two, three or more of copies of nucleic acid sequencesencoding crRNAs, wherein the copies comprise the same spacer sequencefor targeting a host cell sequence (eg, a virulence, resistance oressential gene sequence or a sequence of a host CRISPR/Cas systemcomponent that mediates vector adaptation, but which is not said Casnuclease).

58. The system, vector or collection of example 57, wherein the copiesare split between two or more vector CRISPR arrays.

59. The system, vector or collection of any preceding example, whereinthe vector repeats are identical to repeats in a or the host CRISPRarray (eg, each vector repeat has at least 95% sequence identity to ahost repeat).

60. The system, vector or collection of any one of examples 1 to 58,wherein the vector repeats are not identical to repeats in a or the hostCRISPR array.

61. The system, vector or collection of any preceding example,comprising first and second vector CRISPR arrays which are contained inthe same host cell or by the same vector (eg, plasmid or virus or virionor phage or prophage or phagemid).

62. The system, vector or collection of example 61, wherein the firstarray is contained in a first vector and the second array is containedin a second vector which does not contain the first array (eg, whereinthe vectors are plasmids or virions (eg, of the same virus type) orphagemids (eg, of the same phage type).

63. A host cell comprising a system, vector, collection, virus, virion,phage, phagemid or prophage according to any preceding example.

64. An antimicrobial composition (eg, an antibiotic, eg, a medicine,disinfectant or mouthwash), comprising a system, vector, virus, virion,phage, phagemid or prophage according to any one of examples 1 to 62.

Conditioning Microbes Together

The invention provides for methods of producing microbes (eg, phageand/or bacterial populations) that involves conditioning hosts andviruses together to facilitate co-evolution and thus conditioning of thehosts to the viruses (eg, phage) and vice versa. Using repressiblecontrol of crRNA expression or activity the invention purposelymodulates the co-evolution in a controllable manner where a desiredspacer activity can be toggled on or off to enable tuning to occur withor without stress imposed by spacer-guided Cas action in the host, eg,with or without antibiotic resistance gene targeting. In this way, thebacterial populations can be tuned for use in situations (eg, dairy orfood production cultures) where phage inactivation of desirable genesmay be encountered; or for use in tuning phage to be used to kill ormodulate bacteria, eg, to knock-down antibiotic resistance. Thisconfiguration further enables, in one embodiment, culturing ofantibiotic-resistant bacterial host with virus, eg, phage, harbouringone or more CRISPR arrays of the invention that target the antibioticresistance gene of the host, since the method purposely represses theantibiotic resistance gene inactivation activity of the array duringculturing with the host. Thus, a resistant bacterial host population canbe used to grow up phage in culture (eg, in an industrial culture vesselor plant) allowing the phage and host to co-evolve and mutually tunewithout the antibiotic resistance inactivation effect hampering thegrowth and thus culturing ability of the host cells (which wouldotherwise minimise phage expansion) and whilst still enabling all othercomponents of the desired phage to tune to the cultured host population.Testing of a sample of the resultant phage population can be carriedout, eg, at lab scale, using an antibiotic resistant host cellpopulation but with the test phage de-repressed for the array targetingof the antibiotic resistance gene of the host cells. Naturally-occurringand synthetic repression of gene expression in prokaryotic cell andphage settings is well known to the skilled person, eg, tet systems orlight-inducible systems.

Thus, the invention provides the following features, numbered asparagraphs:—

1. A microbe production method, the method comprising

(a) providing a host cell that comprises a host CRISPR/Cas system fornucleotide sequence targeting in the host cell;(b) providing a virus that is capable of infecting the host cell,wherein(i) the virus comprises one or more engineered host modifying (HM)CRISPR arrays (eg, an array as described above) for modifying targetnucleotide sequences of the host cell;(ii) a first said HM-array encodes a first HM-crRNA comprising a spacersequence (HM-spacer) that is capable of hybridising to a first hosttarget sequence to guide Cas to the target in the host cell to modifythe target sequence, optionally wherein the modification of the firsttarget sequence reduces host cell growth or viability; and(iii) the first HM-array is reversibly repressible for the transcriptionof the first HM-crRNA and/or first HM-crRNA activity is repressible;(c) infecting the host cell with the virus to introduce the one or moreHM-CRISPR arrays into the cell;(d) repressing the transcription of the first HM-crRNA and/or firstHM-crRNA activity in the cell;(e) culturing the infected host cell to produce a population (PH1) ofhost cells comprising a population (PV1) of virus; and(f) obtaining the virus population PV1 and/or the cultured host cellpopulation.

In an example, the first HM-crRNA comprises a HM-spacer that is capableof hybridising to the first host target sequence to guide Cas to thetarget in the host cell to modify the target sequence, wherein thetarget sequence is a nucleotide sequence of the host CRISPR/Cas system,whereby the first HM-crRNA guides Cas to the target to modify the hostCRISPR/Cas system in the host cell, wherein the modification of thetarget sequence reduces or eliminates functioning of the host CRISPR/Cassystem.

In an alternative, the modification enhances or inhibits expression of agene in the host. In an embodiment, the gene is an essential gene,virulence gene or resistance gene (eg, an antibiotic resistance gene).In an embodiment, the modification enhances the expression of a geneproduct that is endogenous or exogenous to the host. In an example, thehost is an engineered host comprising an exogenous nucleotide sequence(eg, for producing a desired protein) and the modification enhances orinhibits expression of the desired protein in the host cell. In anexample, the desired protein is an antibiotic and host cell is amicrobe, eg, bacterial or archaeal cell. Thus, the method enablesculturing of culturing of host cells to produce the viral population,wherein the antibiotic is not expressed which would otherwise hamper theexpansion of the host cell population. Thereafter, one or more virusesof the isolated virus population can be used in an antimicrobialcomposition for reducing host cell growth or viability, since the firstHM-crRNA repression can be removed after isolation, thereby providing anactively antibiotic virus composition. The invention therefore alsoprovides such a method and such an antibiotic composition comprisingvirus that are capable of expressing an antibiotic in a host cell.Modification to activate the expression can be effected, for example, byproviding a Cas (eg, Cas9) conjugated to a transcription activator,wherein the Cas is a cognate Cas for the first HM-crRNA and theactivator activates the transcription of the desired exogenous orendogenous gene. Modification to inhibit the expression can be effected,for example, by providing a dead Cas (eg, dCas9), wherein the CAs is acognate Cas for the first HM-crRNA and inhibits transcription of thedesired exogenous or endogenous gene.

Repression of the crRNA transcription or activity can be partial orcomplete (ie, no activity or no transcription of the crRNA from thearray in the host). Activity refers to the ability of the crRNA tohybridise to the cognate host sequence for guiding of Cas to the firsthost target site for modification.

In an example, the virus is not so repressed when introduced into thecell, the method comprising carrying out step (d) after the virus hasinfected the cell, eg, by using a chemical, physical, mechanical,magnetic, light or other agent to cause repression. In an embodiment,the first HM-array comprises a repressible promoter (HM-promoter) fortranscription of the first HMcrRNA and the promoter is repressed (eg, bybinding a repressor agent, eg, a chemical or protein, to the promoter)after the first HM-array is introduced into the cell.

In another example, the virus is so repressed before step (c) is carriedout, eg, by using a chemical, physical, mechanical, magnetic, light orother agent to cause repression. In an embodiment, the first HM-arraycomprises a repressible promoter (HM-promoter) for transcription of thefirst HMcrRNA and the promoter is repressed (eg, by binding a repressoragent, eg, a chemical or protein to the promoter) before the firstHM-array is introduced into the cell, wherein subsequently the repressedfirst HM-array is introduced into the cell.

In one embodiment, step (f) comprises isolating PV1. In an embodiment,the step comprised separating PV1 or a virus thereof from host cells ofPH1.

2. The method of paragraph 1, further comprising de-repressing thetranscription of first HM-crRNA and/or first HM-crRNA activity in thevirus population after step (e) or (f), and optionally thereafterfurther culturing the host cells.

3. The method of any preceding paragraph, comprising

A. obtaining a population (PH2) of host cells that are optionallyidentical to the host cell of (a), (f) or the further cultured cells ofparagraph 2;B. infecting the host cells of A with virus from the population PV1;C. repressing the transcription of the first HM-crRNA and/or firstHM-crRNA activity in the cells;D. culturing the infected host cells to produce a population (PH3) ofhost cells comprising a population of virus (PV2); andE. obtaining the virus population PV2 (or a virus thereof) and/or thecultured host cell population.

4. The method of paragraph 3, further comprising de-repressing thetranscription of first HM-crRNA and/or first HM-crRNA activity in thevirus population after step (D) or (E), and optionally thereafterfurther culturing the host cells.

5. The method of any preceding paragraph, comprising testing an isolatedsample of the virus population PV1 or PV2 on a further host cell orpopulation (PH4) of host cells, optionally wherein the further cell orpopulation PH4 is identical to the cell of (a), the testing comprisinginfecting the further cell or population PH4 with virus of said sample,waiting a period of time to allow any host cell growth to occur, anddetermining if a predetermined activity of the further cell orpopulation PH4 (eg, cell growth or viability) has been modified (eg,reduced, such as reduced host cell growth or viability*) or occurred,wherein virus inside the cell or cells have de-repressed transcriptionof first HM-crRNA and/or first HM-crRNA activity during said period oftime. * This can be tested using a standard assay for plaque formationwhen the virus of the sample are added to the cell or PH4 plated onagar).

6. The method of any preceding paragraph 5, wherein all of the hostcells are microbial cells (eg, bacterial or archaeal cells) and themodification of the first target sequence reduces host cell growth orviability, and said determining determines that antimicrobial activity**has occurred. **This can be determined using a standard plaque assay.

7. The method of paragraph 5 or 6, wherein the period of time is atleast one, 5, 10, 30, 60 or 120 minutes.

8. The method of any one of paragraphs 5 to 7, wherein the cell of (a)and optionally PH1, PH2 and/or PH3 cells do not comprise the firsttarget sequence, wherein the further cell or population PH4 cellscomprise the first target sequence.

9. The method of any one of paragraphs 1 to 8, wherein the cell of (a)and optionally PH1, PH2 and/or PH3 cells do not comprise a gene thatconfers resistance to a first antibiotic, wherein the first targetsequence is a target sequence of such a gene; optionally wherein thefurther cell or population PH4 cells comprise said gene.

10. The method of any one of paragraphs 1 to 7, wherein the cell of (a)and optionally PH1, PH2 and/or PH3 cells comprise a gene that confersresistance to a first antibiotic, wherein the first target sequence is atarget sequence of such a gene.

11. The method of any preceding paragraph, wherein all of the host cellsare microbial cells (eg, bacterial or archaeal cells) and themodification of the first target sequence reduces host cell growth orviability, or reduces host cell resistance to an antibiotic.

12. The method of any preceding paragraph, wherein all of the host cellsare infectious disease pathogens of humans, an animal (eg, non-humananimal) or a plant.

13. The method of any preceding paragraph, wherein all of the host cellsare of the same species, eg, selected from a species of Escherichia (eg,E coli O157:H7 or O104: H4), Shigella (eg, dysenteriae), Salmonella (eg,typhi or enterica, eg, serotype typhimurium, eg, DT 104), Erwinia,Yersinia (eg, pestis), Bacillus, Vibrio, Legionella (eg, pneumophilia),Pseudomonas (eg, aeruginosa), Neisseria (eg, gonnorrhoea ormeningitidis), Bordetella (eg, pertussus), Helicobacter (eg, pylori),Listeria (eg, monocytogenes), Agrobacterium, Staphylococcus (eg, aureus,eg, MRSA), Streptococcus (eg, pyogenes or thermophilus), Enterococcus,Clostridium (eg, dificile or botulinum), Corynebacterium (eg,amycolatum), Mycobacterium (eg, tuberculosis), Treponema, Borrelia (eg,burgdorferi), Francisella, Brucella, Campylobacter (eg, jejuni),Klebsiella (eg, pneumoniae), Frankia, Bartonella, Rickettsia,Shewanella, Serratia, Enterobacter, Proteus, Providencia, Brochothrix,Bifidobacterium, Brevibacterium, Propionibacterium, Lactococcus,Lactobacillus, Pediococcus, Leuconostoc, Vibrio (eg, cholera, eg, O139,or vulnificus), Haemophilus (eg, influenzae), Brucella (eg, abortus),Franciscella, Xanthomonas, Erlichia (eg, chaffeensis), Chlamydia (eg,pneumoniae), Parachlamydia, Enterococcus (eg, faecalis or faceim, eg,linezolid-resistant), Oenococcus and Acinetoebacter (eg, baumannii, eg,multiple drug resistant).

14. The method of claim 13, wherein all of the host cells areStaphylococcus aureus cells, eg, resistant to an antibiotic selectedfrom methicillin, vancomycin-resistant and teicoplanin.

15. The method of claim 13, wherein all of the host cells arePseudomonas aeuroginosa cells, eg, resistant to an antibiotic selectedfrom cephalosporins (eg, ceftazidime), carbapenems (eg, imipenem ormeropenem), fluoroquinolones, aminoglycosides (eg, gentamicin ortobramycin) and colistin.

16. The method of claim 13, wherein all of the host cells are Klebsiella(eg, pneumoniae) cells, eg, resistant to carbapenem.

17. The method of claim 13, wherein all of the host cells areStreptococcus (eg, pneumoniae or pyogenes) cells, eg, resistant to anantibiotic selected from erythromycin, clindamycin, beta-lactam,macrolide, amoxicillin, azithromycin and penicillin.

18. The method of claim 13, wherein all of the host cells are Salmonella(eg, serotype Typhi) cells, eg, resistant to an antibiotic selected fromceftriaxone, azithromycin and ciprofloxacin.

19. The method of claim 13, wherein all of the host cells are Shigellacells, eg, resistant to an antibiotic selected from ciprofloxacin andazithromycin.

20. The method of claim 13, wherein all of the host cells aremycobacterium tuberculosis cells, eg, resistant to an antibioticselected from Resistance to isoniazid (INH), rifampicin (RMP),fluoroquinolone, amikacin, kanamycin and capreomycin.

21. The method of claim 13, wherein all of the host cells areEnterococcus cells, eg, resistant to vancomycin.

22. The method of claim 13, wherein all of the host cells areEnterobacteriaceae cells, eg, resistant to an antibiotic selected from acephalosporin and carbapenem.

23. The method of claim 13, wherein all of the host cells are E. colicells, eg, resistant to an antibiotic selected from trimethoprim,itrofurantoin, cefalexin and amoxicillin.

24. The method of claim 13, wherein all of the host cells areClostridium (eg, dificile) cells, eg, resistant to an antibioticselected from fluoroquinolone antibiotic and carbapenem.

25. The method of claim 13, wherein all of the host cells are Neisseriagonnorrhoea cells, eg, resistant to an antibiotic selected from cefixime(eg, an oral cephalosporin), ceftriaxone (an injectable cephalosporin),azithromycin and tetracycline.

26. The method of claim 13, wherein all of the host cells areAcinetoebacter baumannii cells, eg, resistant to an antibiotic selectedfrom beta-lactam, meropenem and a carbapenem.

27. The method of claim 13, wherein all of the host cells areCampylobacter cells, eg, resistant to an antibiotic selected fromciprofloxacin and azithromycin.

28. The method of any preceding paragraph, wherein the host cellsproduce Beta ((3)-lactamase.

29. The method of any preceding paragraph, wherein the host cells areresistant to an antibiotic recited in any one of paragraphs 14 to 27.

30. The method of paragraph 29, wherein the first target sequence is asequence of a gene encoding a product conferring host cell resistance tosaid antibiotic.

31. The method of any preceding paragraph, wherein the first targetsequence is a sequence of an antibiotic resistance gene (ie, forconferring host cell resistance to an antibiotic eg, methicillinresistance) and/or one, more or all of the population PH1, thepopulation PH2, the population PH3 and the population PH4 is resistantto an antibiotic or said antibiotic (eg, an antibiotic recited in anyone of paragraphs 13 to 27).

32. The method of any preceding paragraph, wherein de-repressed virus ofthe virus population PV1 or PV2 have antimicrobial activity (eg,antibacterial activity, such as when the virus are phage); optionallywherein the host cell or cells comprise the first target sequence asrecited in paragraph

30, wherein modification of the first target provides said antimicrobialactivity.

33. The method of any preceding paragraph when dependant from paragraph5, wherein the cells of PH4 are resistant to an antibiotic (eg, anantibiotic recited in any one of paragraphs 13 to 27) and the cells of(a) and PH2 are not resistant to said antibiotic. This aidsmanufacturing of the virus for drug use, since culturing and expansioncan be performed relatively safety without the risk of having to dealwith antibiotic-resistant host cells (and risk of inadequate containmentof these and escape from drug manufacturing plant, for example).Nevertheless, testing against PH4 can be performed in a containment labor other facility that is set up for use of antibiotic-resistant hoststrains. When testing against PH4, the first HM-crRNA is de-repressed sothat modification of the resistance gene in the host cells is possibleby the HM-array of the invention.

34. The method of any preceding paragraph, wherein the host CRISPR/Cassystem is a Type I, II or III system and the target sequence is anucleotide sequence conserved in said Type of system in at least one,two or three additional host strains or species of the same genus as thehost cell of (a).

35. The method of any preceding paragraph, wherein the virus is a phageor phagemid.

36. The method of paragraph 35, wherein the virus of (b) is aCorticoviridae, Cystoviridae, Inoviridae, Leviviridae, Microviridae,Myoviridae, Podoviridae, Siphoviridae, or Tectiviridae virus.

37. The method of paragraph 35 or 36, wherein the virus of (b) is anaturally occurring phage, eg, a phage induced from a cell that is ofthe same strain as the cell of (a).

38. The method of paragraph 35, 36 or 37, wherein the phage of (b) is amutated phage obtained through selective pressure using aphage-resistant bacterium.

39. The method of any preceding paragraph, wherein in (b)

(iv) said one or more HM-arrays comprise a HM-array that encodes asecond HM-crRNA comprising a HM-spacer that is capable of hybridising toa second host target sequence to guide Cas to the second target in thehost cell to modify the target sequence, wherein the second targetsequence is a nucleotide sequence of the host CRISPR/Cas system, wherebythe second HM-crRNA guides Cas to the second target to modify the hostCRISPR/Cas system in the host cell, wherein the modification of thesecond target sequence reduces or eliminates functioning of the hostCRISPR/Cas system; and(v) wherein the HM-array of (iv) is active in the cell of (a) for thetranscription of second HM-crRNA capable of hybridising to the secondhost target sequence.

In an embodiment, the HM-array of (ii) and (iv) are the same HM-array.In another embodiment, they are different HM-arrays (eg, arrays ofdifferent CRISPR/Cas types, eg, Type I and II, or Type II and III, orType I and III, or different Type II arrays).

40. The method of paragraph 39, wherein the cells of any one or all ofPH1-4 comprise said second target sequence.

41. The method of paragraph 39 or 40, wherein the second target sequenceis identical to a CRISPR/Cas system sequence of a genus or species ofcell as recited in any one of paragraphs 11 to 24 (eg, S thermophilus orS pyogenes or S aureus).

42. The method of any one of paragraphs 39 to 41, wherein the secondtarget sequence is comprised by a sequence selected from the groupconsisting of SEQ ID NO: 1 to 44, or a complement thereof.

43. The method of any one of paragraphs 39 to 42, wherein the secondtarget sequence comprises

A. a repeat DNA or RNA sequence (eg, wherein the repeat is the 5′-mostrepeat (the first repeat) in said host CRISPR array;B. a tracrRNA sequence or a tracrRNA-encoding DNA sequence; a CRISPRarray leader sequence;C. a Cas gene promoter (eg, a Cas1, Cas2 or Csn2 promoter);D. a CRISPR array leader promoter sequence; orE. a Cas-encoding DNA or RNA sequence (eg, wherein the Cas is Cas9,Cas1, Cas2 or Csn2).

44. The method of any one of paragraphs 39 to 43, wherein the secondtarget sequence comprises

F. a CRISPR array leader or leader promoter sequence contiguous with the5′-most nucleotide of the first repeat (and optionally comprising said5′-most nucleotide of the repeat);G. a sequence of up to 20 contiguous nucleotides immediately 5′ of thefirst repeat;H. a sequence of up to 20 contiguous nucleotides of the 5′-mostnucleotides of the first repeat; orI. a sequence of up to 20 contiguous nucleotides immediately 3′ of thefirst spacer repeat (and optionally wherein the sequence comprises the3′-most nucleotide of the first spacer).

45. The method of any one of paragraphs 39 to 44, wherein

J. the second HM-crRNA comprises or consists of the structure R—S—R,wherein R=a CRISPR repeat and S=a CRISPR spacer, wherein S comprises,(in 5′ to 3′ direction) V-H_(R) or H_(R)—V or, wherein V=a sequence atleast 95, 96, 97, 98 or 99% identical to a DNA sequence of the virus of(b) and H_(R)=a DNA sequence of a CRISPR repeat of said host cellCRISPR/Cas system;K. wherein the sequence of H_(R) is immediately contiguous with thesequence of V in the host CRISPR/Cas system; andL. wherein the second HM-crRNA is capable of hybridising to a spacer ofthe host CRISPR/Cas system to guide Cas to the spacer for modification(eg, cleavage or inactivation) of the host CRISPR/Cas system in thecell.

46. The method of paragraph 45, wherein V=one or up to 40 (eg, up to 15)contiguous nucleotides of virus DNA.

47. The method of any one of paragraphs 39 to 46, wherein the secondHM-crRNA does not substantially hybridise to nucleic acid of the virusof (b).

48. The method of any one of paragraphs 45 to 47, wherein

a. the host CRISPR/Cas system is able to recognise a cognate PAM;b. wherein the nucleic acid of the virus of (b) comprises such a PAMimmediately 3′ of a protospacer sequence;c. wherein V=one or up to 40 (eg, up to 15) nucleotides of theprotospacer; andd. wherein H_(R)=a sequence identical to a contiguous sequence of therepeat of the host CRISPR/Cas system.

49. The method of paragraph 48, wherein said contiguous sequence of therepeat of the host system is a sequence of at least 50% of a host repeat(eg, including the 5′-most or 3′-most nucleotide of the host repeat).

50. The method of paragraph 45 or 46, wherein V=from 1 to 40 (eg, up to15) of the 3′-most protospacer contiguous nucleotides; and optionallysaid contiguous sequence of the repeat includes the 5′-most nucleotideof the host repeat.

51. The method of paragraph 48 or 49, wherein V=from 1 to 40 (eg, up to15) of the 5′-most protospacer contiguous nucleotides; and optionallysaid contiguous sequence of the repeat includes the 3′-most nucleotideof the host repeat.

52. The method of any one of paragraphs 45 to 51, wherein R=a repeatthat is recognised by the host CRISPR/Cas system.

53. The method of any preceding paragraph, wherein the or each HM-CRISPRcomprises (in

5′ to 3′ direction) a first repeat sequence, a first spacer sequence anda second repeat sequence, wherein the spacer sequence comprises asequence that is capable of hybridising to the respective targetsequence in the host cell, the array further comprising a promoter fortranscription of the repeats and spacer in the host cell, and optionallythe nucleic acid of the virus of (b) comprises a Cas nuclease-encodingsequence and/or a tracrRNA-encoding sequence for encoding a functionalCas and/or tracrRNA sequence in the host cell, wherein the tracrRNAsequence comprises a sequence that is complementary to the first orsecond repeat.

54. The method of any preceding paragraph, wherein the or each HM-CRISPRarray comprises (in 5′ to 3′ direction) a first repeat sequence, a firstspacer sequence and a second repeat sequence, wherein the spacersequence comprises a sequence that is capable of hybridising to therespective target sequence in the host cell, the array furthercomprising a promoter for transcription of the repeats and spacer in thehost cell, and wherein the vector does not comprise a Casnuclease-encoding sequence and/or a tracrRNA-encoding sequence forencoding a tracrRNA sequence in the host cell wherein the tracrRNAsequence comprises a sequence that is complementary to the first orsecond repeat, wherein the HM-CRISPR array is functional in the hostcell to guide Cas (eg, endogenous host Cas nuclease) to the respectivehost target site, optionally using a host tracrRNA.

55. The method of paragraph 53 or 54, wherein the repeats are identicalto repeats in the host CRISPR/Cas system, wherein the or each HM-CRISPRarray does not comprise a PAM recognised by a Cas (eg, a Cas nuclease,eg, Cas9) of the host CRISPR/Cas system.

56. The method of any preceding paragraph, wherein the or each HM-CRISPRarray comprises more than one copy of a HM-spacer (eg, at least 2, 3 or4 copies).

57. The method of any preceding paragraph, encoding a second or thirdHM-crRNA (further HM-crRNA), wherein the further HM-crRNA comprises anucleotide sequence that is capable of hybridising to a host targetsequence to guide Cas to the target in the host cell; optionally whereinthe target sequence is a nucleotide sequence of an essential, virulenceor resistance gene of the host cell, or of an essential component of theCRISPR/Cas system of the host cell.

58. The method of any preceding paragraph, wherein the or each HM-CRISPRarray comprises CRISPR repeat sequences that are identical to endogenousCRISPR repeat sequences of the host cell for producing the respectiveHM-crRNA in the host cell.

59. The method of any preceding paragraph, wherein the virus of (b)comprises a nucleotide sequence encoding a Cas (non-host Cas) that isfunctional in the host cell of (a) (eg, wherein the non-host Cas is aType I system Cas wherein the host system is a Type II or III; a Type IIsystem Cas wherein the host system is a Type I or III; or a Type IIIsystem Cas wherein the host system is a Type I or II), optionallywherein the host cell does not comprise or express a Cas of a Type thatis the same as the Type of the non-host Cas.

60. The method of any preceding paragraph, wherein the virus of (b)comprises a nucleotide sequence encoding a tracrRNA sequence, optionallywherein the tracrRNA sequence and first HM-crRNA are comprised by asingle guide RNA (gRNA)).

61. The method of any preceding paragraph, wherein the or each HM-crRNAis comprised by a respective single guide RNA (gRNA).

62. The method of any preceding paragraph, wherein the first HM-array isoperable to cause Cas cleavage in the first target sequence, activationof the first target sequence (or gene comprising the first targetsequence), knock-down of the first target sequence (or gene comprisingthe first target sequence) or mutation of the first target sequence.

63. A virus, host cell or virus population obtainable by the method ofany preceding paragraph, optionally wherein the population is identicalto PV1 or PV2 or the virus is obtainable from such a population.

64. A host cell (eg, bacterial cell) population obtainable by the methodof any preceding paragraph, optionally wherein the population isidentical to PH1, PH2, PH3 or PH4 or a cultured cell population recitedin any preceding paragraph.

65. The host cell population of paragraph 64 wherein the population doesnot comprise nucleic acid of a virus of (b), or does not comprise saidfirst HM-array or said second HM-array (eg, as determined by PCR).

66. The virus, host cell or population of any one of paragraphs 63 to65, for medical or dental or ophthalmic use (eg, for treating orpreventing an infection in an organism or limiting spread of theinfection in an organism.

67. A composition comprising a virus, host cell or population accordingto any one of paragraphs 63 to 66 for food, beverage, dairy or cosmeticuse (eg, use in a cosmetic product, eg, make-up), or for hygiene use(eg, use in a hygiene product, eg, soap).

68. Use of a composition a virus, host cell or population according toany one of paragraphs 63 to 67, in medicine or for dental therapeutic orprophylactic use.

69. Use of a composition a virus, host cell or population according toany one of paragraphs 63 to 68, in cosmetic use (eg, use in a cosmeticproduct, eg, make-up), or for hygiene use (eg, use in a hygiene product,eg, a soap).

70. The use, virus, host cell or population of any one of paragraphs 63to 69 for modifying a microbial host cell (eg, for killing or reducinggrowth of the cell or a culture of microbe cells).

71. The method, virus or virus population of any one of paragraphs 1 to63 and 66 to 70, wherein the virus or virus in said population express aholin and/or an endolysin for host cell lysis, optionally wherein theendolysin is a phage phi11, phage Twort, phage P68, phage phiWMY orphage K endolysin (eg, MV-L endolysin or P-27/HP endolysin).

72. The method, virus or virus population of any one of paragraphs 1 to63 and 66 to 70, wherein the virus or virus in said population does noexpress a holin and/or an endolysin for host cell lysis.

73. The method, virus or virus population of any one of paragraphs 1 to63 and 66 to 70, wherein the virus (eg, virus of (b)) or virus in eachsaid population is in combination with an antimicrobial functional inthe host cell of (a), eg, antibiotic agent, eg, a beta-lactam antibiotic(eg, an antibiotic recited in any one of paragraphs 13 to 27).

Control of Corrosion, Biofilms & Biofouling

The invention relates inter alia to methods of controllingmicrobiologically influenced corrosion (MIC) or biofouling of asubstrate or fluid in an industrial or domestic system. The inventionalso relates to treated fluids and vectors for use in the methods.

Corrosion is the result of a series of chemical, physical and (micro)biological processes leading to the deterioration of materials such asmetal (eg, steel or iron), plastic and stone. It is a worldwide problemwith great societal and economic consequences. Current corrosion controlstrategies based on chemically produced products are under increasingpressure of stringent environmental regulations. Furthermore, they arerather inefficient and may be hampered by microbial (eg, bacterial)resistance to the agents used. Therefore, there is an urgent need forenvironmentally friendly and sustainable corrosion control strategies.Corrosion is influenced by the complex processes of differentmicroorganisms performing different electrochemical reactions andsecreting proteins and metabolites that can have secondary effects.

The severity of microbial corrosion processes is evident from the factthat many of the industrially and domestically used metals and alloyssuch as stainless steels, nickel and aluminium-based alloys andmaterials such as concrete, asphalt and polymers are readily degraded bymicroorganisms. Protective coatings, inhibitors, oils and emulsions arealso subject to microbial degradation.

Microbially influenced corrosion (MIC) is a costly problem that impactshydrocarbon production and processing equipment, water distributionsystems, ships, railcars, and other types of metallic and non-metallicindustrial and domestic systems. In particular, MIC is known to causeconsiderable damage to hydrocarbon fuel infrastructure includingproduction, transportation, and storage systems, oftentimes withcatastrophic environmental contamination results. Around 40% of pipecorrosion in the oil industry is attributed to microbiological corrosionand leads to huge financial losses in production, transportation andstorage of oil every year. Pipe biofilms can cause the reduction influid velocity in equipment due to the process of incrustation on walls.Furthermore, pipe leaks are generated as a result of the corrosion, withconsequent impacts on the environment and productivity.

MIC takes place in environments such as soil, fresh water and sea waterand is estimated to be responsible for more than 30 percent of allcorrosion damage. MIC occurs due to the fixation of microbes such asbacteria, release of metabolites and usually formation of biofilms thatinduce or accelerate the corrosion process. Among the groups of bacteriainvolved in the corrosion process are included: sulphur- orsulphate-reducing bacteria (SRB), extracellular polymericsubstance-producing bacteria (EPSB), acid-producing bacteria (APB),sulphur- or sulphide-oxidising bacteria (SOB); iron- ormanganese-oxidising bacteria (IOB), ammonia producing bacteria (AmPB)and acetate producing bacteria (AcPB). Small subunit ribosomal RNA genepyrosequencing surveys indicate that acetic-acid-producing bacteria(Acetobacter spp. and Gluconacetobacter spp.) are prevalent inenvironments exposed to fuel-grade ethanol and water.

Microbial growth under environmental conditions influenceselectrochemical reactions directly or indirectly. Microbe-substrateinteractions lead to initial adhesion and biofilm formation. Theattachment of microbes such as bacteria to substrate, release ofmetabolites and formation of biofilms influences the electrochemicalconditions at substrate surfaces, inducing or accelerating the corrosionprocess, thereby mediating the process of MIC. The formation of abacterial biofilm on a metallic substrate comprises the followingstages: I—formation of a film, through the adsorption of organic andinorganic molecules on the metal, which modifies the load distributionon the metallic surface and, also serves as a nutritional source for thebacteria, facilitating the adherence of free-floating microorganismspresent in the liquid; II—adhesion and multiplication of aerobicbacteria forming microcolonies; III—production of extracellularpolymeric substances (EPS) by some sessile bacteria; IV—colonisation byaerobic free-floating microbial cells, that will consume the oxygen byrespiration, creating a local anaerobic environment in the biofilm asrequired by strict anaerobic bacteria and; V—increase of biofilmthickness, which may favour the shedding of the outer layers. The EPSproduced by the bacteria adhered to the biofilm capture essential ionsfor their growth; they are used as a means of attachment and protectbacteria against biocides interfering with the mechanisms of corrosionby favouring the creation of differential aeration areas, besidesserving as a nutritional source in case of low nutrient availability.The process of corrosion by differential aeration occurs due to unevendistribution of the biofilm on the metal substrate with aerated regions(surrounding the biofilm) and non-aerated regions (below the biofilm).The biofilm formation on the metal surface decreases the oxygen content,reaching levels of almost total anaerobiosis. Pseudomonas is the mainEPS producer genus.

An example of a MIC biocorrosion process mediated by corrosive bacteriais as follows: (A) Aerobic corrosive bacteria from fresh water, seawater, industrial/domestic systems or storage tanks reach out equipmentand pipelines of industrial or domestic systems, that have aconditioning film on the surface. (B) EPS-producing bacteria attach toequipment/pipeline walls and produce EPS, which creates a favourableenvironment for adhesion by other microorganisms. (C) Adhesion of othergroups of corrosive bacteria to pipeline walls takes place, whichrelease their metabolites, developing into a microcolony through celldivision, consuming oxygen available. Action of iron-oxidising bacteriaresults in a large accumulation of ferric precipitation leading toblockage in the equipment/pipeline; sulphuric acid released bysulphur-oxidising bacteria promotes the acidification of theenvironment. (D) The low oxygen concentration and organic acids releasedby acid-producing bacteria favour attachment and development ofsulphate-reducing bacteria producing hydrogen sulphide (H₂S), therebyaccelerating the corrosion process and reducing the local pH. (E) Acorroded equipment/pipeline results, which is partially blocked by ironprecipitates with micro-leaks and containing a bacterial biofilm. TheH₂S poses a serious health risk to personnel operating the systemaffected. Furthermore, the production of thick biofilms and sludges leadto biofouling and hampering of the functioning of the system. Similarly,bacterial populations may propagate in fluids, such as water stores orreservoirs (eg, in drinking water or in water of cooling systems),thereby mediating biofouling of the fluid. This may also be referred toas souring of the fluid. An example is waterway or drinking waterreservoir souring.

The invention addresses such problems of MIC and biofouling by providingthe following Aspects 1 et seq:—

1. A method of controlling microbiologically influenced corrosion (MIC)or biofouling of a substrate in an industrial or domestic system,wherein a surface of the substrate is in contact with a population offirst host cells of a first microbial species that mediates MIC orbiofouling of the substrate, the method comprising

(i) contacting the population with a plurality of vectors that arecapable of transforming or transducing the cells, each vector comprisinga CRISPR array whereby CRISPR arrays are introduced into the host cells,wherein(a) each CRISPR array comprises one or more nucleotide sequences forexpression of a crRNA and a promoter for transcription of thesequence(s) in a host cell; and(b) each crRNA is capable of hybridising to a target sequence of a hostcell to guide Cas (eg, a Cas nuclease, eg, a Cas9 or Cpf1) in the hostcell to modify the target sequence (eg, to cut the target sequence); thetarget sequence being a gene sequence for mediating host cell viability;and(ii) allowing expression of said cRNAs in the presence of Cas in hostcells, thereby modifying target sequences in host cells, resulting inreduction of host cell viability and control of MIC or biofouling ofsaid substrate.

In an example, the system comprises equipment (eg, for use in anindustrial process) and the surface is a surface of said equipment. Inan example, each array is an engineered array, eg, any engineered arraydisclosed herein. In an embodiment, the vector is an engineered CRISPRnucleic acid vector as described herein. In an example, the biofoulingcomprises microbial biofilm and/or sludge formation, proliferation ormaintenance. In an example, the first host cells are sessile. In anexample of Aspect 1 or 4 (below), “controlling” comprises preventing,reducing or eliminating said MIC or biofouling, or reducing spread ofsaid MIC or biofouling in the system. Non-limiting examples of howbacteria mediate MIC or biofouling are described above. Cell growth orproliferation or maintenance is, for example, a characteristic of cellviability. Thus, in an example, the method reduces host cellproliferation and/or maintenance. In an example, the method kills hostcells.

2. The method of Aspect 1, wherein said host cells are comprised by amicrobial biofilm that is in contact with said substrate.

3. The method of any preceding Aspect, wherein said surface and hostcells are in contact with a fluid, such as an aqueous liquid (eg, seawater, fresh water, stored water or potable water).

Fresh water is naturally occurring water on the Earth's surface in icesheets, ice caps, glaciers, icebergs, bogs, ponds, lakes, rivers andstreams, and underground as groundwater in aquifers and undergroundstreams. Fresh water is generally characterized by having lowconcentrations of dissolved salts and other total dissolved solids. Theterm specifically excludes sea water and brackish water, although itdoes include mineral-rich waters such as chalybeate springs. In anexample said fresh water is any of these fresh water types. Potablewater is water for human or animal (eg, livestock) consumption.

In an example, the fluid is selected from industrial cooling waterwherein the system is a cooling system; sewage water wherein the systemis a sewage treatment or storage system; drinking water wherein thesystem is a drinking water processing, storage, transportation ordelivery system; paper making water wherein the system is a papermanufacture or processing system; swimming pool water wherein the systemis a swimming pool or swimming pool water treatment or storage system;fire extinguisher water wherein the system is a fire extinguishingsystem; or industrial process water in any pipe, tank, pit, pond orchannel.

4. A method of controlling microbial biofouling of a fluid in anindustrial or domestic system (eg, for controlling bacterial souring ofa liquid in a reservoir or container), wherein the fluid comprises apopulation of first host cells of a first microbial species thatmediates said biofouling, the method comprising

(i) contacting the population with a plurality of vectors that arecapable of transforming or transducing the cells, each vector comprisinga CRISPR array whereby CRISPR arrays are introduced into the host cells,wherein

(a) each CRISPR array comprises one or more sequences for expression ofa crRNA and a promoter for transcription of the sequence(s) in a hostcell; and

(b) each crRNA is capable of hybridising to a target sequence of a hostcell to guide Cas (eg, a Cas nuclease) in the host cell to modify thetarget sequence (eg, to cut the target sequence); the target sequencebeing a gene sequence for mediating host cell viability; and

wherein the method comprises allowing expression of said cRNAs in thepresence of Cas in host cells, thereby modifying target sequences inhost cells, resulting in reduction of host cell viability and control ofsaid biofouling.

In an example, the fluid is a liquid. In an example, the fluid is agaseous fluid.

Systems:

An example system for any Aspect is selected from the group consistingof a:—Petrochemical recovery, processing, storage or transportationsystem; hydrocarbon recovery, processing, storage or transportationsystem; crude oil recovery, processing, storage or transportationsystem; natural gas recovery, processing, storage or transportationsystem, (eg, an oil well, oil rig, oil drilling equipment, oil pumpingsystem, oil pipeline, gas rig, gas extraction equipment, gas pumpingequipment, gas pipeline, oil tanker, gas tanker, oil storage equipmentor gas storage equipment); Water processing or storage equipment; waterreservoir (eg, potable water reservoir); Air or water conditioning (eg,cooling or heating) equipment, eg, a coolant tube, condenser or heatexchanger; Medical or surgical equipment; Environmental (eg, soil,waterway or air) treatment equipment; Paper manufacturing or recyclingequipment; Power plant, eg, a thermal or nuclear power plant; Fuel (eg,hydrocarbon fuel, eg, petroleum, diesel or LPG) storage equipment;Mining or metallurgical, mineral or fuel recovery system, eg, a mine ormining equipment; Engineering system; Shipping equipment; Cargo or goodsstorage equipment (eg, a freight container); Food or beveragemanufacturing, processing or packaging equipment; Cleaning equipment(eg, laundry equipment, eg, a washing machine or dishwasher); Catering(eg, domestic or commercial catering) equipment; Farming equipment;Construction (eg, building, utilities infrastructure or roadconstruction) equipment; Aviation equipment; Aerospace equipment;Transportation equipment (eg, a motor vehicle (eg, a car, lorry or van);a railcar; an aircraft (eg, an aeroplane) or a marine or waterwayvehicle (eg, a boat or ship, submarine or hovercraft)); Packagingequipment, eg, consumer goods packaging equipment; or food or beveragepackaging equipment; Electronics (eg, a computer or mobile phone or anelectronics component thereof); or electronics manufacture or packagingequipment; Dentistry equipment; Industrial or domestic piping (eg, asub-sea pipe) or storage vessel (eg, a water tank or a fuel tank (eg,gasoline tank, eg, a gasoline tank of a vehicle)); Undergroundequipment; Building (eg, a dwelling or office or commercial premises orfactory or power station); Roadway; Bridge; Agricultural equipment;Factory system; Crude oil or natural gas exploration equipment; Officesystem; and a Household system.

In an example, the system is used in an industry or business selectedfrom the group consisting of agriculture, oil or petroleum industry,food or drink industry, clothing industry, packaging industry,electronics industry, computer industry, environmental industry,chemical industry, aerospace industry, automotive industry,biotechnology industry, medical industry, healthcare industry, dentistryindustry, energy industry, consumer products industry, pharmaceuticalindustry, mining industry, cleaning industry, forestry industry, fishingindustry, leisure industry, recycling industry, cosmetics industry,plastics industry, pulp or paper industry, textile industry, clothingindustry, leather or suede or animal hide industry, tobacco industry andsteel industry. In an example, the surface or fluid to be treated is asurface or fluid of equipment used in said selected industry. In anexample, the system is used in the crude oil industry. In an example,the system is used in the natural gas industry. In an example, thesystem is used in the petroleum industry. In an example, the system is asea container, platform or rig (eg, oil or gas platform or rig for useat sea or at sea), ship or boat. In an embodiment, such a system isanchored at sea; eg, non-temporarily anchored at sea, eg, has beenanchored at sea for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24 or more months (eg, contiguousmonths). In an embodiment, such a system is in the waters of a countryor state; eg, non-temporarily at sea in such waters, eg, has been inwaters of said country for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more months (eg,contiguous months).

In an example, the substrate surface to be treated comprises stainlesssteel, carbon steel, copper, nickel, brass, aluminium, concrete, aplastic or wood. In an example, the substrate is a metal weld or join.In an example, the surface is a metallic (eg, steel or iron) ornon-metallic (eg, plastic, concrete, asphalt, wood, rubber or stone)surface. In an example, the metal is an alloy (eg, stainless steel,brass or a nickel-, zinc-, copper-, nickel- or aluminium-alloy). In anexample, the surface is a man-made polymer surface. In an example, thesurface is a substrate coating. In an example, the substrate is incontact with soil, fresh water or sea water.

In an example, the fluid is potable water; a waterway; brackish water;or a liquid fuel, eg, gasoline or diesel (eg, for a car or motorizedvehicle), LPG, kerosine, an alcohol (eg, ethanol, methanol or butanol),liquid hydrogen or liquid ammonia), in an example, the fuel is storedliquid fuel. In an example the fluid is an oil or non-aqueous liquid. Inan example, the fluid is a liquid comprised by a waterway or body ofwater, eg, sea water, fresh water, potable water, a river, a stream, apond, a lake, a reservoir, stored water (eg, in a water storage tank orcooling equipment), groundwater, well water, water in a rock formation,soil water or rainwater. In an example, the liquid is sea water. In anexample, the substrate is in contact with a liquid mentioned in thisparagraph. In an example, the fluid or liquid is selected from the groupconsisting of an oil, an aqueous solution, a hydraulic fracturing fluid,a fuel, carbon dioxide, a natural gas, an oil/water mixture, afuel/water mixture, water containing salts, ocean or sea water, brackishwater, sources of fresh water, lakes, rivers, stream, bogs, ponds,marshes, runoff from the thawing of snow or ice, springs, groundwater,aquifers, precipitation, any substance that is a liquid at ambienttemperature (eg, at rtp) and is hydrophobic but soluble in organicsolvents, hexanes, benzene, toluene, chloroform, diethyl ether,vegetable oils, petrochemical oils, crude oil, refined petrochemicalproducts, volatile essential oils, fossil fuels, gasoline, mixtures ofhydrocarbons, jet fuel, rocket fuel, biofuels. In an example the fluidis an oil/water mixture.

The terms “microbiologically influenced corrosion” or “MIC” as usedherein, unless otherwise specified, refer to processes in which anyelement (substrate) of a system is structurally compromised due to theaction of at least one member of a microbial population, eg, bacterialor archaeal population. The term “biofouling” as used herein, unlessotherwise specified, refers to processes in which microorganisms (suchas bacteria and/or archaea) accumulate on a substrate surface in contactwith a fluid (eg, water or an aqueous liquid, or a hydrocarbon, or apetrochemical). Also included is the undesirable accumulation andproliferation of microorganisms (such as bacteria and/or archaea) in afluid (eg, water or an aqueous liquid, or a hydrocarbon, or apetrochemical), ie, “souring” of the fluid. In an example, the bacteriaare comprised by ship or boat ballast water and the bacteria areenvironmentally undesirable. The term “substrate” as used herein refersto any type of surface on which cells can attach and a biofilm can formand grow or on which biofouling (eg slime or sludge formation) canoccur. The substrate may be an “industrial” substrate such as thesurface of equipment in an petrochemical, fuel, crude oil or gas pipingsystem, or a “non-industrial” (eg, domestic, eg, household or office)substrate such as a kitchen counter or a shower substrate or a gardensubstrate.

In an alternative of any of the Aspects, instead of a population of hostbacterial cells, the population is a population of archaeal cells of afirst species.

5. The method of Aspect 4, wherein said fluid is an aqueous liquid (eg,sea water, fresh water, stored water or potable water).

6. The method of any one of Aspects 3 to 5, wherein the method comprisesmixing the fluid with the vectors, thereby contacting the host cellswith vectors. For example, the vectors can be pre-mixed with a liquid(optionally with an antibiotic or biocide too) and the mixture thenadded to the fluid that is in contact with the surface (Aspect 1) or thefluid of Aspect 4.

7. The method of any one of Aspects 1-6, wherein each target sequence isa host cell virulence, resistance or essential gene sequence, eg, anexon or regulatory sequence thereof. Resistance can be antibioticresistance. In an example, the host cells are contacted with saidantibiotic and said vectors to reduce host cell viability.

8. The method of any one of Aspects 1-7, wherein the modification oftarget sequences results in host cell killing and/or a reduction in hostcell growth or proliferation. Proliferation is, for example, cellexpansion or cell distribution in contact with the surface.

9. The method of any one of Aspects 1-8, wherein the vectors compriseidentical CRISPR arrays.

10. The method of any one of Aspects 1-9, wherein the host cells arebacterial or archaeal cells. In an alternative, instead the first cellsare algal cells.

11. The method of any one of Aspects 1-10, wherein the first host cellsare sulphate reducing bacteria (SRB) cells (eg, Desulfovibrio orDesulfotomaculum cells). In an example, the cells are selected from thegroup consisting of Desulfotomaculum nigrificans, Desulfacinum infernum,Thermodesulfobacterium mobile, Thermodesulforhabdus norvegicus,Archaeoglobus fulgidus, Desulfomicrobium apsheronum, Desulfovibriogabonensis, Desulfovibrio longus, Desulfovibrio vietnamensis,Desulfobacterium cetonicum, Desulphomaculum halophilum, Desulfobactervibrioformis and Desulfotomaculum thermocisternum cells. In an example,the population comprises a mixture of two or more of these cell species.

12. The method of Aspect 11, wherein the surface or fluid is comprisedby a crude oil, gas or petrochemicals recovery, processing, storage ortransportation equipment. Crude oil is one of the most importantenergetic resources in the world. It is used as raw material in numerousindustries, including the refinery-petrochemical industry, where crudeoil is refined through various technological processes into consumerproducts such as gasoline, oils, paraffin oils, lubricants, asphalt,domestic fuel oil, vaseline, and polymers. Oil-derived products are alsocommonly used in many other chemical processes. In an alternative, thefluid is a said consumer product or the surface is in contact with sucha consumer product.

13. The method of Aspect 11 or 12, wherein the surface is in contactwith sea water, a fracking liquid or liquid in a well; or wherein thefluid is sea water, a fracking liquid or liquid in a well.

14. The method of any one of Aspects 1-13, wherein step (i) of themethod comprises providing a population of microbial cells of a secondspecies (second host cells), the second cells comprising said vectors,wherein the vectors are capable of transfer from the second host cellsto the first host cells; and combining the second host cells with thefirst host cells, whereby vectors are introduced into the first hostcells. In an example, the second cell(s) are environmentally-,industrially-, or domestically-acceptable in an environment (eg, in awater or soil environment) and the first host cell(s) are not acceptablein the environment.

15. The method of 14, wherein the first host cells are comprised by amixture of microbial cells (eg, comprised by a microbial biofilm) beforecontact with said vectors, wherein the mixture comprises cells of saidsecond species.

16. The method of Aspect 14 or 15, wherein said second species is aspecies of Bacillus or nitrate-reducing bacteria or nitrate reducingsulfide oxidizing bacteria (NRB).

17. The method of Aspect 16, wherein the NRB is selected from the groupconsisting of Campylobacter sp., Nitrobacter sp., Nitrosomonas sp.,Thiomicrospira sp., Sulfurospirillum sp., Thauera sp., Paracoccus sp.,Pseudomonas sp., Rhodobacter sp. and Desulfovibrio sp; or comprises atleast 2 of said species.

18. The method of Aspect 17 wherein NRB is selected from the groupconsisting of Nitrobacter vulgaris, Nitrosomonas europea, Pseudomonasstutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans,Sulfurospirillum deleyianum, and Rhodobacter sphaeroides.

19. The method of any one of Aspects 1-18, wherein the method comprisescontacting the host cells of said first species with a biocidesimultaneously or sequentially with said vectors. In an example, thevectors and biocide are provided pre-mixed in a composition that iscontacted with the host cells.

20. The method of Aspect 19, wherein the biocide is selected from thegroup consisting of tetrakis hydroxymethyl phosphonium sulfate (THPS),glutaraldehyde, chlorine monoxide, chlorine dioxide, calciumhypochlorite, potassium hypochlorite, sodium hypochlorite,dibromonitriloproprionamide (DBNPA), methylene bis(thiocyanate) (MBT),2-(thiocyanomethylthio) benzothiazole (TCMTB), bronopol,2-bromo-2-nitro-1,3-propanediol (BNPD), tributyl tetradecyl phosphoniumchloride (TTPC), taurinamide and derivatives thereof, phenols,quaternary ammonium salts, chlorine-containing agents, quinaldiniumsalts, lactones, organic dyes, thiosemicarbazones, quinones, carbamates,urea, salicylamide, carbanilide, guanide, amidines, imidazolines, aceticacid, benzoic acid, sorbic acid, propionic acid, boric acid,dehydroacetic acid, sulfurous acid, vanillic acid, p-hydroxybenzoateesters, isopropanol, propylene glycol, benzyl alcohol, chlorobutanol,phenylethyl alcohol, formaldehyde, iodine and solutions thereof,povidone-iodine, hexamethylenetetramine, noxythiolin,1-(3-chloroallyl)-3,5,7-triazo-1-azoniaadamantane chloride, taurolidine,taurultam, N-(5-nitro-2-furfurylidene)-1-amino-hydantoin,5-nitro-2-furaldehyde semicarbazone, 3,4,4′-trichlorocarbanilide,3,4′,5-tribromosalicylanilide,3-trifluoromethyl-4,4′-dichlorocarbanilide, 8-hydroxyquinoline,1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylicacid,1,4-dihydro-1-ethyl-6-fluoro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylicacid, hydrogen peroxide, peracetic acid, sodium oxychlorosene,parachlorometaxylenol, 2,4,4′-trichloro-2′-hydroxydiphenol, thymol,chlorhexidine, benzalkonium chloride, cetylpyridinium chloride, silversulfadiazine, silver nitrate, bromine, ozone, isothiazolones,polyoxyethylene (dimethylimino) ethylene (dimethylimino) ethylenedichloride, 2-(tert-butylamino)-4-chloro-6-ethylamino-5′-triazine(terbutylazine), and combinations thereof. In an example the biocide istetrakis hydroxymethyl phosphonium sulfate (THPS). In an example, thebiocide is a quaternary ammonium compound.

21. The method of any one of Aspects 1-20, wherein the system is used inan industry operation selected from the group consisting of mining;shipping; crude oil, gas or petrochemicals recovery or processing;hydraulic fracturing; air or water heating or cooling; potable waterproduction, storage or delivery; transportation of hydrocarbons; andwastewater treatment.

22. The method of Aspect 21, wherein the surface is a surface ofequipment used in said selected industry; or wherein the fluid is afluid comprised by equipment used in said selected industry.

23. The method of any one of Aspects 1-22, wherein the surface is asurface of kitchen, bathing or gardening equipment; or wherein the fluidis comprised by kitchen, bathing or gardening equipment. For example,the equipment is used in a domestic setting.

24. The method of any one of Aspects 1-23 when dependent from Aspect 3,wherein the fluid is a potable liquid contained in a container (eg,water tank or bottle) and the surface is a surface of the container incontact with the liquid.

25. The method of any one of Aspects 1-24, wherein each vector comprisesa mobile genetic element (MGE), wherein the MGE comprises an origin oftransfer (oriT) and a said CRISPR array; wherein the MGE is capable oftransfer between a host cell of said first species and a furthermicrobial host cell in said industrial or domestic system. For example,the further cell(s) are environmentally-, industrially-, ordomestically-acceptable in an environment (eg, in a water or soilenvironment) and the first host cell(s) are not acceptable in theenvironment.

26. The method of Aspect 25, wherein oriT is functional in the first andfurther host cells.

27. The method of Aspect 25 or 26, wherein said first and further hostcells are comprised by a biofilm of fluid in contact with said surface;or wherein said cells are comprised by said fluid.

28. The method of Aspect 25, 26 or 27, wherein said further cell is acell of a species as recited in any one of Aspects 16 to 18. In anexample, the MGE is capable of transfer from the further cell to thefirst host cell and/or vice versa.

29. The method of any one of Aspects 25 to 27, wherein the further cellis a cell of said first species.

For example, in this embodiment the MGE is capable of transfer amongstfirst cells in a population in said system. When the MGE leaves a copyof itself in the transfer process to the other cell, this then providesmeans for propagating and spreading the MGE and thus CRISPR arraysthrough cell populations in the system, thereby spreading the targetsequence modifying effect of the arrays. This can be effective, forexample, to create spread of arrays in a biofilm in contact with thesurface or in the fluid, and is useful as penetration of biofilms withconventional biocides can be sub-optimal.

30. The method of any one of Aspects 25 to 29, wherein each MGE is orcomprises an integrative and conjugative element (ICE); or wherein eachvector is a phage that is capable of infecting host cells of said firstspecies and each MGE is a phage nucleic acid that is capable of saidtransfer between the cells.

31. The method of Aspect 30, wherein each ICE is a transposon, eg, aconjugative transposon.

32. The method of any one of Aspects 1-31, wherein each vector is aplasmid, optionally comprising an MGE according to any one of Aspects 25to 31.

33. The method of any one of Aspects 25 to 32, wherein the first and/orfurther cell comprises nucleotide sequences encoding proteins operableto transfer the MGE to the other cell, wherein the sequences are notcomprised by the MGE.

34. The method of Aspect 33, wherein the sequences are not comprised bythe vector.

35. The method of Aspect 33, wherein the sequences are comprised by aconjugative transposon of the first cell and/or further cell.

36. The method of Aspect 35, wherein the transposon is operable in transto transfer the MGE between the first and further cells.

37. The method of any one of Aspects 25 to 36, wherein the oriT of theMGE is the same as an oriT comprised by an ICE of the first cell and/orfurther cells, wherein the ICE is operable in trans to transfer the MGEbetween the first and further cells.

38. The method of any one of Aspects 25 to 37, wherein the vector oriTis an oriT of a SRB or NRB transposon.

39. The method of any one of Aspects 25 to 38, wherein each MGEcomprises first and second terminal repeat sequences and a said CRISPRarray between the repeat sequences.

40. The method of any one of Aspects 25 to 39, wherein the MGE leavesbehind a CRISPR array copy (1) in the genome of a first host cell whenit has transferred to a said further host cell; or (2) in a said furtherhost cell when it has transferred to a first host cell. For example, thecopy is comprised by a transposon or prophage left in the genome of thecell from which transfer takes place.

41. The method of any one of Aspects 25 to 40, wherein the first andfurther cells are bacterial cells of different species (eg, SRB and NRB;or SRB and Bacillus cells respectively).

42. The method of any one of Aspects 25 to 41 when dependent from Aspect30 in combination with a transposase for mobilisation of the MGE.

43. The method of any one of Aspects 1-42, wherein the vector or MGEcomprises a toxin-antioxin module that is operable in a host cell ofsaid first species; optionally wherein the toxin-antitoxin modulecomprises an anti-toxin gene that is not operable or has reducedoperation in cells of another species. These embodiments are useful tocreate a selective pressure that favours retention of the vector/MGE(and thus CRISPR arrays) in the first host cells comprising the targetsequences.

44. The method of any one of Aspects 1-43, wherein the vector or MGEcomprises a toxin-antioxin module that is operable in a said second orfurther cell; optionally wherein the toxin-antitoxin module comprises ananti-toxin gene that is not operable or has reduced operation in cellsother than the second or further cell. This is useful to maintain apopulation of CRISPR arrays in the second or further cells (eg, whensuch cells are present in a biofilm also comprising the first cells),but wherein the toxin-antitoxin module provides additional killing (overand above the action of the target sequence modification) in first hostcells. In an example, the vector or MGE comprises a toxin-antioxinmodule that is operable in a first host cell and in said second orfurther cell.

45. The method of any one of Aspects 43 or 44, wherein thetoxin-antitoxin module is not operable or has reduced operation in cellsother than the first and second or further cells. Thus, there can be aselective pressure in both the first and second (or further) cells tomaintain the CRISPR arrays. Usefully, this then provides a reservoir forhorizontal transfer of the arrays in MGEs between cells in a mixedpopulation (eg, a biofilm contacting the surface or a populationcomprised by the fluid).

46. The method of any one of Aspects 25-45 wherein the first and secondcells (or first and further cells) are of the same phylum (eg, bothbacterial cells) and the vector is replicable or operable (A) in thefirst cell and/or second (or further) cell but not in another cell ofthe same phylum; (B) in the first cell and/or second (or further) cellbut not in another cell of the same order; (C) in the first cell and/orsecond (or further) cell but not in another cell of the same class; (D)in the first cell and/or second (or further) cell but not in anothercell of the same order; (E) in the first cell and/or second (or further)cell but not in another cell of the same family; (F) in the first celland/or second (or further) cell but not in another cell of the samegenus; or (G) in the first cell and/or second (or further) cell but notin another cell of the same species.

47. The method of Aspect 25 or any one of Aspects 26 to 46 whendependent from Aspect 25, wherein each MGE is a conjugative transposon,oriT is functional in the first and further (or second) host cells, theMGE comprises first and second terminal repeat sequences and a saidCRISPR array between the repeat sequences, and wherein the first andfurther (or second) cells are bacterial cells, wherein the target siteis comprised by the first cells but not the further (or second) cells,and wherein said modifying inactivates or down-regulates a gene orregulatory sequence comprising said target in the first cells, resultingin reduction of first host cell viability and control of said MIC orbiofouling.

48. The method of any one of Aspects 1-47, wherein each CRISPR arraycomprises a sequence R1-S1-R1′ for expression and production of therespective crRNA in a first host cell,

(i) wherein R1 is a first CRISPR repeat, R1′ is a second CRISPR repeat,and R1 or R1′ is optional; and(ii) S1 is a first CRISPR spacer that comprises or consists of anucleotide sequence that is 95% or more identical to a target sequenceof a said first host cell.

49. The method of Aspect 48, wherein R1 and R1′ are at least 95, 96, 97,98 or 99% identical respectively to the first and second repeatsequences of a CRISPR array of the first host cell species. In anembodiment, both R1 and R1′ are present.

50. The method of Aspect 48 or 49, wherein R1 and R1′ are functionalwith a CRISPR/Cas system of said host cells of said first species formodification of target sequences.

51. The method of any one of Aspects 48 to 50, wherein the first hostcells are sulphate reducing bacteria (SRB) cells and R1 and R1′ areleast 95, 96, 97, 98 or 99% identical respectively to a repeat sequence(eg, the first repeat) of a CRISPR array of the first host cell species.

52. The method of Aspect 51, wherein R1 and R1′ are least 95, 96, 97, 98or 99% identical respectively to a repeat sequence selected from thegroup consisting of SEQ ID NOs: 50-74, 125-128 and 49. See Table 1. Inan embodiment, both R1 and R1′ are present.

53. The method of Aspect 51, wherein R1 and R1′ are least 95, 96, 97, 98or 99% identical respectively to a repeat sequence selected from thegroup consisting of SEQ ID NOs: 51 and 125-126, 54 and 127, 69 and 128.SEQ ID NOs: 51 and 125-126, 54 and 127, 69 and 128 are found in morethan one SRB species. This is particularly useful for targeting morethan one SRB type with the CRISPR array of the invention, eg, when theSRB types co-exist in the industrial or domestic system to be treated,for example co-existing in a population or biofilm that is in contactwith the substrate or in the fluid to be treated. In an embodiment, bothR1 and R1′ are present.

54. The method of any one of Aspects 48 to 53, wherein the sequences ofR1 and R1′ are identical.

55. The method of any one of Aspects 1-54, wherein each array introducedinto a first host cell is introduced in combination with one or more Casnuclease(s) (eg, a Cas9 and/or Cfp1) that function with the respectivecrRNA in a host cell to modify a target sequence thereof.

In an example, Cas herein in any configuration is deactivated fornuclease activity and optionally comprises a target sequence activatoror depressor. A Cas 9 herein is, for example S pyogenes or S aureusCas9.

56. The method of any one of Aspects 1-55, wherein each array introducedinto a first host cell is introduced in combination with nucleic acidsequence(s) encoding one or more Cas nuclease(s) (eg, a Cas9 and/orCfp1) that function with the respective crRNA in a host cell to modifythe target sequence.

57. The method of any one of Aspects 48 to 56, wherein R1 and R1′ arefunctional with a Type II Cas9 nuclease to modify a target sequence in asaid first host cell, optionally wherein the method is further accordingto Aspect 55 or 56 wherein the Cas is said Cas9.

58. The method of any one of Aspects 1-57, wherein all or some of saidvectors or MGEs do not comprise a Cas nuclease-encoding sequenceoperable with the respective array.

59. The method of Aspect 58, wherein each said respective array isoperable with a Cas endonuclease found in cells of the first species.

60. The method of Aspect 25, or any one of Aspects 26 to 59 whendependent from Aspect 25, wherein each MGE is devoid of a sequenceencoding a Cas endonuclease that is operable with repeat sequences ofthe array, and wherein the respective vector comprises such a sequence(eg, encoding a Cas9 of Cfp1) outside the MGE.

61. A method of controlling microbiologically influenced corrosion (MIC)or biofouling of a substrate comprised by a crude oil, gas orpetrochemicals recovery, processing, storage or transportation equipment(eg, a crude oil tanker, oil rig or oil drilling equipment), wherein asurface of the substrate is in contact with a population of first hostcells, wherein the first host cells are sulphur- or sulphate-reducingbacteria (SRB), extracellular polymeric substance-producing bacteria(EPSB), acid-producing bacteria (APB), sulphur- or sulphide-oxidizingbacteria (SOB), iron-oxidising bacteria (IOB), manganese-oxidisingbacteria (MOB), ammonia producing bacteria (AmPB) or acetate producingbacteria (AcPB) of a first species that mediates MIC or biofouling ofthe substrate, wherein the surface and cell population are in contactwith a liquid selected from sea water, fresh water, a fracking liquid orliquid in a well (eg, oil or natural gas well), the method comprising

(i) contacting the cell population with vectors by mixing the liquidwith a plurality of vectors that are capable of transforming ortransducing first host cells, each vector comprising a CRISPR arraywhereby CRISPR arrays are introduced into the host cells, wherein

(a) each CRISPR array comprises one or more sequences for expression ofa crRNA and a promoter for transcription of the sequence(s) in a hostcell;

(b) each crRNA is capable of hybridising to a target sequence of a hostcell to guide Cas (eg, a Cas nuclease, eg, a Cas9 or Cfp1) in the hostcell to modify the target sequence (eg, to cut the target sequence); thetarget sequence being a gene sequence for mediating host cell viability;

(c) wherein each sequence of (a) comprises a sequence R1-S1-R1′ forexpression and production of the respective crRNA in a first host cell,wherein R1 is a first CRISPR repeat, R1′ is a second CRISPR repeat, andR1 or R1′ is optional; and S1 is a first CRISPR spacer that comprises orconsists of a nucleotide sequence that is 70, 75, 80, 85, 90 or 95% ormore identical to a target sequence of a said first host cell and

(ii) allowing expression of said cRNAs in the presence of Cas in hostcells, thereby modifying target sequences in host cells, resulting inreduction of host cell viability and control of MIC or biofouling ofsaid substrate. In an embodiment, both R1 and R1′ are present.

62. The method of Aspect 61, wherein the method is according to Aspect 1or any preceding Aspect when dependent from Aspect 1.

63. The method of Aspect 61 or 62, wherein each vector is a phagecapable of infecting a first host cell or is a vector comprising a MGE(eg, a transposon) that comprises a said CRISPR array, wherein the MGEis capable of transfer into a first host cell.

64. The method of Aspect 61, 62 or 63, wherein the first cells aresulphate reducing bacteria (SRB) cells, eg, Desulfovibrio orDesulfotomaculum cells.

65. The method of Aspect 64, wherein R1 and R1′ are at least 95, 96, 97,98 or 99% identical respectively to a repeat sequence (eg, the firstrepeat) of a CRISPR array of the first host cell species and the vectorarrays are operable with a Cas endonuclease found in cells of the firstspecies. In an example, R1 and R1′ are identical sequences.

66. The method of Aspect 65, wherein R1 and R1′ are at least 95, 96, 97,98 or 99% identical respectively to a repeat sequence selected from thegroup consisting of SEQ ID NOs: 50-74, 125-128 and 49. In an example, R1and R1′ are identical sequences.

67. The method of Aspect 66, wherein R1 and R1′ are at least 95, 96, 97,98 or 99% identical respectively to a repeat sequence selected from thegroup consisting of SEQ ID NOs: 51 and 125-126, 54 and 127, 69 and 128.See Table 1. This is particularly useful for targeting more than one SRBtype with the CRISPR array of the invention, eg, when the SRB typesco-exist in the industrial or domestic system to be treated, for exampleco-existing in a population or biofilm that is in contact with thesubstrate or in the fluid to be treated. In an example, R1 and R1′ areidentical sequences.

68. The method of any one of Aspects 1-67, wherein said plurality ofvectors comprise additional vectors, wherein each additional vectorcomprises one or more CRISPR arrays for targeting additional host cellscomprised by said population, wherein the additional host cell speciesis different from the first host cell species, wherein in step (i) saidadditional cells of the population are contacted with a plurality ofsaid additional vectors that are capable of transforming or transducingthe additional cells, each vector comprising a CRISPR array wherebyCRISPR arrays are introduced into the additional host cells, wherein

(a) each CRISPR array comprises one or more sequences for expression ofa crRNA and a promoter for transcription of the sequence(s) in a hostcell; and

(b) each crRNA is capable of hybridising to a target sequence of a saidadditional host cell to guide Cas (eg, a Cas nuclease) in the host cellto modify the target sequence (eg, to cut the target sequence); thetarget sequence being a gene sequence for mediating host cell viability;and step (ii) comprises allowing expression of said cRNAs in thepresence of Cas in said additional host cells, thereby modifying targetsequences in additional host cells.

69. The method of Aspect 68, wherein the additional host cells mediateMIC or biofouling of said substrate or fluid, wherein step (ii) resultsin reduction of additional host cell viability and control of MIC orbiofouling of said substrate or fluid.

70. A method of controlling bacterial biofouling in ballast water of aship or boat, wherein the water comprises a population of first hostcells of a first microbial species that mediates said biofouling, themethod comprising

(i) contacting the population with a plurality of vectors that arecapable of transforming or transducing the cells, each vector comprisinga CRISPR array whereby CRISPR arrays are introduced into the host cells,wherein

(a) each CRISPR array comprises one or more sequences for expression ofa crRNA and a promoter for transcription of the sequence(s) in a hostcell; and

(b) each crRNA is capable of hybridising to a target sequence of a hostcell to guide Cas (eg, a Cas nuclease) in the host cell to modify thetarget sequence (eg, to cut the target sequence); the target sequencebeing a gene sequence for mediating host cell viability; and

(ii) allowing expression of said cRNAs in the presence of Cas in hostcells, thereby modifying target sequences in host cells, resulting inreduction of host cell viability and control of said biofouling.

71. The method of Aspect 70, wherein the first host cells are Vibriocholerae, E coli or Enterococci sp cells.

72. The method of Aspect 70 or 71, wherein step (i) comprises mixing theballast water with the vectors, eg, in the hull of a ship or boat.

73. The method of any one of Aspects 70 to 72, wherein the ship or boatis a marine vehicle and the water is sea water.

74. The method of any one of Aspects 70 to 72, wherein instead of a shipor boat, the ballast water is comprised by a container or a drillingplatform at sea, eg, an oil platform or oil rig. In an example, theship, boat, container, platform or rig is anchored at sea (ie, nottemporarily in its location).

75. A method of discharging ballast water from a ship or boat, whereinthe discharged ballast water comprises water treated by the method ofany one of Aspects 70 to 74.

76. The method of Aspect 75, wherein the water is discharged into a bodyof water, eg, a sea, ocean or waterway (eg, a river, canal, lake orreservoir) or into a container.

77. Ballast sea water comprising CRISPR arrays, wherein the ballastwater is obtained or obtainable by the method of any one of Aspects 70to 76.

78. A ship, boat, container or rig comprising the ballast sea water ofAspect 77.

79. A vector for use in the method of any one of Aspects 61 to 69,wherein the first cells are sulphate reducing bacteria (SRB) cells, eg,Desulfovibrio or Desulfotomaculum cells, each vector comprising one ormore CRISPR arrays for targeting the SRB, wherein each array is asdefined in (a)-(c) of Aspect 61.

80. The vector of Aspect 79, wherein R1 and R1′ are according to any oneof Aspects 65 to 67.

81. A vector for use in the method of any one of Aspects 70 to 76,wherein the first cells are Cholera (eg, vibrio, eg, O1 or O139), E colior Enterococci sp cells, the vector comprising one or more CRISPR arraysfor targeting the cells, wherein each array is as defined in (a) and (b)of Aspect 70.

82. The vector of any one of Aspects 79 to 81, wherein the vector is abacteriophage capable of infecting a said cell.

83. The vector of any one of Aspects 79 to 81, wherein the vector is atransposon or MGE capable of transfer into a said cell.

84. A plurality vectors, wherein each vector is according to Aspect 82or 83, optionally in combination with a biocide or antibiotic that iscapable of reducing viability of said cells.

Bacteria that Mediate MIC or Biofouling:

In an example, the first host cells are selected from the groupconsisting of sulphur- or sulphate-reducing bacteria (SRB),extracellular polymeric substance-producing bacteria (EPSB, eg,Pseudomonas), acid-producing bacteria (APB), sulphur- orsulphide-oxidising bacteria (SOB); iron- or manganese-oxidising bacteria(IOB), ammonia producing bacteria (AmPB) and acetate producing bacteria(AcPB). For example, the first host cells are AcPB (eg, Acetobacter spp.and/or Gluconacetobacter spp) and the surface is in contact with ahydrocarbon fuel (eg, fuel-grade ethanol) and/or water.

The following are examples of relevant bacteria for the presentinvention (in an example, the first host cells are cells of any of thefollowing species). Acidithiobacillus bacteria produce sulphuric acid.Acidithiobacillus thiooxidans, a subgenus of Acidithiobacillus bacteria,frequently damages sewer pipes. Ferrobacillus ferrooxidans directlyoxidises iron to iron oxides and iron hydroxides. Other bacteria producevarious acids, both organic and mineral, or ammonia. In the presence ofoxygen, aerobic bacteria like Thiobacillus thiooxidans, Thiobacillusthioparus, and Thiobacillus concretivorus, all three widely present inthe environment, are the common corrosion-causing factors resulting inbiogenic sulphide corrosion. Without presence of oxygen, anaerobicbacteria, especially Desulphovibrio and Desulphotomaculum, are common.Desulphovibrio salixigens requires at least 2.5% concentration of sodiumchloride, but D. vulgaris and D. desulphuricans can grow in both freshand salt water. D. africanus is another common corrosion-causingmicroorganism. The Desulphotomaculum genus comprises sulphate-reducingspore-forming bacteria. Desulphotomaculum orientis and nigrificans areinvolved in corrosion processes. Sulphate-reducers require a reducingenvironment, and an electrode potential of at least −100 mV is requiredfor them to thrive. However, even a small amount of produced hydrogensulphide can achieve this shift, so the growth, once started, tends toaccelerate.

In an Example the first host cells are Serratia marcescens, Gallionellasp., Pseudomonas sp., Bacillus sp. (eg, B. subtilis, B. cereus, B.pumilus or B. megaterium), Thiobacillus sp., Sulfolobus sp., Klebsiellaoxytoca, Pseudomonas aeruginosa, P. stutzeri, Micrococcus, Enterococcus,Staphylococcus (eg, S. aureus), E. faecalis or M. luteus cells. In anexample, the first host cells comprise a mixture of two or more of saidspecies. These species have been isolated from diesel andnaphtha-transporting pipelines located in the northwest and southwestregions in India; the association with localized corrosion of thepipeline steel in the presence of these consortia was corroborated. Ajoint project of different european aircraft manufacturers confirmed theinvolvement of isolates from genera Micrococcus, Enterococcus,Staphylococcus and Bacillus in strong corrosion damage in aluminiumalloy, commonly used in aircraft construction. These bacteria may createa microacidic environment (acid producing bacteria), which favours thedevelopment of other bacteria, or produce EPS, favouring the formationof biofilm (EPS-producing bacteria). Thus, in an embodiment of theinvention, the surface (eg, steel surface) of the system to be treatedis in contact with diesel or naptha, or the fluid to be treated isdiesel or naptha (and optionally the first host cells are of one or morespecies defined in this paragraph). In an embodiment of the invention,the surface (eg, aluminium-containing surface, eg, an aircraft surface)of the system to be treated is in contact with one, two, three or allgenera: Micrococcus, Enterococcus, Staphylococcus and Bacillus (firsthost cells). In an example of any embodiment in this paragraph, thesurface is a surface of a steel or aluminium component of the system.

Acid-Producing Bacteria:

Aerobic bacteria are able to produce short-chain organic acids such asacetic, formic, lactic, propionic and butyric acids as products of theirmetabolism from the fermentative metabolism of organic materials. Theyare also initial colonizers due to aerobic metabolism. Thesemicroorganisms are present in a variety of environments, including gasstands and oils. Organic acids serve as substrates for the SRB,accelerating the corrosion process, besides reducing the pH of thesurrounding medium. Furthermore, the large amount of organic acidproduced acts in metal depolarization, starting the local corrosiveprocess.

Sulphur-Oxidising Bacteria:

The sulphur-oxidising bacteria are aerobic and facultative anaerobicmicroorganisms which obtain the energy necessary for growth from theoxidation of inorganic sulphur compounds such as sulphide, sulphite,thiosulphate and, in some cases the sulphur. Oxidative metabolismresults in the production of sulphuric acid which promotes environmentacidification. This group encompasses many genera, the Acidithiobacillusgenus being the most studied. The group also includes bacterial speciesfrom the genera Sulfolobus, Thiomicrospira, Beggiatoa,Acidithiobacillus, and Thiothrix as well as the species Thiosphaerapantotropha and Paracoccus denitrificans. In an example, the first hostcells are cells of any one of these species.

Iron-Oxidising Bacteria:

Iron oxidising bacteria are aerobic microorganisms, belonging to a largeand diverse group, that get energy necessary for their metabolism fromiron oxidation. Consequently, there is the formation of iron hydroxidesthat generally form insoluble precipitate on substrate surfaces,promoting regions with different oxygen levels. They are widely found inwater from rivers, lakes and oil production. They have mostly alocomotor sheath and their presence can be detected by a largeaccumulation of ferric precipitated as corrosion product. Thisaccumulation or inorganic fouling leads to problems to industrialequipment such as blockages in oil pipelines. Among the most common are:Thiobacillus ferrooxidans and the genera Crenothrix, Gallionella,Leptothrix and Spherotillus. In an example, the first host cells arecells of any one of these species.

Sulphur—or Sulphate-Reducing Bacteria (SRB):

The SRB form a morphological- and phylogenetically heterogenous groupthat includes bacteria and restricted anaerobic archaeabacteria,although some species have significant tolerance to oxygen. They aremainly gram-negative bacteria, mesophilic and some thermophilicgenerally spore-forming. These microorganisms are capable of oxidisingvarious organic compounds of low molecular weight, including mono- ordicarboxylic aliphatic acids, alcohols, hydrocarbons and aromaticcompounds, using sulphate ions or other sulphur compounds (thiosulphate,sulphite, etc.) as electron acceptors. Acetate, lactate, pyruvate andethanol are among the most commonly used substrates by SRB. Thestimulation of SRB growth is due to existing anaerobic conditions inbiofilms explained by the deposition of corrosion products combined withmicroorganisms and, during oil recovery, where there is injection ofaqueous media such as sea water, rich in sulphate. Large amounts ofbiogenic hydrogen sulphide can be produced; most of the H₂S formed inpipelines and other oil, gas or petrochemicals recovery, processing,storage or transportation equipment originates from the metabolicactivity of SRB. Another economic impact on the oil industry is theacidification of oil and gas by H₂S.

Considering the numerous economic losses related to metabolic activityof SRB, efforts have been directed to the use of environmentally-harmfuland toxic metabolic inhibitors such as molybdate, nitrate and nitrite,and application of biocides, which help the control of metabolicactivity of SRB and subsequent inhibition of biogenic H₂S production.

Several mechanisms contribute to contain the formation process ofbiogenic H₂S by using metabolic inhibitors: I—competition between SRBand heterotrophic bacteria that are reducers of nitrite or nitrate byordinary electron donors, resulting in competitive SRB exclusion;II—increased redox potential due to the presence of intermediaries ofnitrate reduction (nitrous oxide and nitric oxide), since the biologicalproduction of H₂S occurs only at low redox potential (below −100 mV);III—Change of energy metabolism of some SRB, reducing nitrate instead ofsulphate; IV—sulphide oxidising bacteria and nitrate or nitrite reducingbacteria that use the nitrate or nitrite to re-oxidise H₂S, resulting inH₂S removal; V—inhibition of the dissimilatory sulphite reductase bynitrite to inhibit the final enzymatic step via sulphate reduction inSRB.

In certain embodiments of the present invention, the host cellpopulation in contact with the substrate to be treated or comprised bythe fluid to be treated is also contacted with one or more nitrateand/or one or more nitrite in the presence of the vectors of theinvention. For example, in step (i) simultaneously or sequentially withthe vectors, the nitrate/nitrite and vectors are combined with (eg,injected into) oil, gas, petrochemical, water or other fluid comprisedby the industrial or domestic system. Similarly, additionally oralternatively, molybdates also may also be used in these systems as acontrol mechanism for SRB. Thus, in one embodiment, the host cellpopulation in contact with the substrate to be treated or comprised bythe fluid to be treated is also contacted with one or more molybdate inthe presence of the vectors of the invention. For example, in step (i)simultaneously or sequentially with the vectors, the molybdate(s) andvectors are combined with (eg, injected into) oil, gas, petrochemical orother fluid comprised by the industrial or domestic system.

In other embodiments, the population is contacted with nitrate-reducingbacteria and/or nitrate reducing sulphide oxidising bacteria (NRSOB)(herein collectively, “NRB”) in the presence of the vectors of theinvention. For example, simultaneously or sequentially with the vectors,the NRB are combined with (eg, injected into) oil, gas, petrochemical,water or other fluid comprised by the industrial or domestic system. Inan example, the NRB comprise vectors of the invention, wherein thevectors are capable of transfer from the NRB cells to the first hostcells (SRB cells); and following combining the NRB and SRB cells, thevectors are introduced into the SRB cells. In an example, the SRB cellsare comprised by a mixture of microbial cells (eg, comprised by amicrobial biofilm) before contact with said vectors, wherein the mixturecomprises cells of the NRB species. Thus, in this case the inventioninvolves contacting the SRB cells with NRB cells (containing vectors)where the NRB cell species are already co-existing with the SRB in thebiofilm to be targeted, which thus increases compatibility and chance ofuptake of the vector-containing NRB into the biofilm cell population.This is useful for increasing the chances of the vectors being takeninto the biofilm, thereby increasing chances of efficacy to modify SRBcells and chances of propagation of the CRISPR arrays of the inventionwithin the biofilm (especially when the arrays are comprised by mobilegenetic elements, such as transposons or comprised by phage, as hereindescribed).

SRB and NRB typically compete for the same non-polymer carbon source(such as acetates) present in certain oilfield and industrial watersystems needed for growth of bacteria. By increasing the growth rate ofthe NRB in comparison to the SRB, the NRB may out compete the SRB inconsumption of the available non-polymer carbon source, depriving theSRB of its ability to grow and create the undesirable sulphides andreduce corrosion rates. Further, by inhibiting the growth rate of theSRB, the NRB may predominate, again out competing the SRB for theavailable non-polymer carbon in the system, eg, oilfield or industrialwater system. Thus, contacting the SRB cells in the population with NRBcan help to reduce SRB cell viability by increasing the ratio of NRB toSRB in the population.

In an embodiment, the invention comprises contacting the populationcomprising the first host cell (eg, SRB) with organic and/or inorganicnitrates and nitrite. These serve to stimulate the growth of the NRBpresent, thus helping the NRB to outcompete SRB. Organic and inorganicnitrates or inorganic nitrites may be used injected into the certainoilfield and industrial water systems. Inorganic nitrates and inorganicnitrites available for use in the present disclosure include, forinstance, potassium nitrate, potassium nitrite, sodium nitrate, sodiumnitrite, ammonium nitrate, and mixtures thereof. These organic andinorganic nitrates and inorganic nitrites are commonly available, butare non-limiting and any appropriate nitrate or nitrite may be used.

The amount of organic or inorganic nitrate or nitrite used is dependentupon a number of factors, including the amount of sulphate and/ororganic acids present in the population in the system, and the expectedamount of NRB needed to counteract the SRB. In certain embodiment, fortreating MIC of a substrate in contact with a liquid, or for treatingbiofouling of a liquid according to the invention, the concentration oforganic or inorganic nitrate or nitrite used is less than 2000 ppm byweight of the liquid, alternatively 500 to 1600 ppm by weight oralternatively between about 900 and 1100 ppm by weight when appliedusing a batch application method. When applied through continuousoperation, the concentration of the organic or inorganic nitrate ornitrite may be less than 500 ppm by weight, alternatively between 10 and500 ppm, or alternatively between 10 and 100 ppm of the liquid.

In an embodiment, the population is contacted with the vectors of theinvention and simultaneously or sequentially with NRB (eg, that comprisethe vectors) and nitrate and/or nitrite.

Suitable NRB include any type of bacteria capable of performinganaerobic nitrate reduction, such as heterotrophic nitrate-reducingbacteria, and nitrate-reducing sulphide-oxidising bacteria. In anexample, the NRB comprises one, two, three or more (eg, one or more) NRBselected from the group consisting of Campylobacter sp. Nitrobacter sp.,Thiobacillus sp., Nitrosomonas sp., Thiomicrospira sp., Sulfurospirillumsp., Thauera sp., Paracoccus sp., Pseudomonas sp. and Rhodobacter sp.For example, the NRB is selected from one or more of Nitrobactervulgaris, Nitrosomonas europea, Pseudomonas stutzeri, Pseudomonasaeruginosa, Paracoccus denitrificans, Sulfurospirillum deleyianum, andRhodobacter sphaeroides.

In certain embodiments, the NRB is a NRB strain that is found in a crudeoil, gas, petrochemical or water recovery, processing, transportation orstorage system (eg, in equipment thereof), or is found in a subterraneanformation, such as a water or oil well. The NRB may be optimized tometabolize under the system conditions. The NRB are, for example,selected from a library of NRB strains or may be cultured from thesystem to be treated or a similar system.

The amount of NRB contacted with the SRB cells in the system may dependupon a number of factors including the amount of SRB expected, as wellas any biocide that may be present. When injected into subterraneanformation, the permeability and porosity of the subterranean formationmay be considered as well. In certain embodiments of the presentdisclosure, the amount of NRB injected into the liquid is between 1 and10⁸ bacteria count/ml of the liquid, or alternatively between 10 and 10⁴bacteria count/ml of the liquid.

In addition to stimulating the NRB to out compete the SRB, it may bedesirable to introduce additional SRB inhibitors in certain embodimentsof the present disclosure together with the nitrates. In an example, theSRB are contacted with one or more SRB inhibitors selected from thegroup consisting of 9,10-anthraquinone, molybdates (such as sodiummolybdate and/or lithium molybdate) and mixtures thereof. In certainembodiments of the present disclosure, molybdate is added to the liquidin the range of 5 to 100 ppm by weight of liquid.

In an example, vectors of the invention and one or more biocides (ie,biocides of the first host cells, such as SRB biocides) are mixed priorto contacting the first cells with the mixture, eg, by injection of themixture into liquid that is in contact with the surface to be treated orinjection of the mixture into the fluid to be treated.

Additionally or alternatively to NRB cells containing vectors, theinvention contemplates use of a species of Bacillus cells comprisingvectors of the invention.

In an embodiment, the vectors are bacteriophage that are capable ofinfecting the SRB and the phage are contacted with the first host cells(eg, SRB), whereby CRISPR arrays comprised by the phage are introducedinto first host cells for modification thereof according to theinvention. In an embodiment, when the first cells are SRB, the SRB arealso contacted with the phage vectors of the invention andsimultaneously or sequentially with NRB. Instead of, or in addition tocontacting with NRB, the SRB are contacted with nitrate and/or nitrite.

Example mechanisms involved in MIC are as follows; in an embodiment, the“controlling” using the method comprises reducing a mechanism selectedfrom:

Microbial (eg, bacterial) promotion of bio-mineralisation due todeposition of iron hydroxides on the metal surface, modifying theelectrochemical processes at the interface metal/solution, inducingcorrosion;

Production of EPS that favours the formation of biofilm;

Microbial (eg, bacterial) promotion of the degradation of petroleumproducts due to the release of the enzyme aryl hydrocarbon hydroxylase(AHH) that acts on the corrosion of metals;

Production of sulphuric acid, which increases the corrosion process; and

Oxidation of sulphur.

In an embodiment, the method comprises reducing a mechanism selectedfrom:

Bacterial promotion of bio-mineralisation due to deposition of ironhydroxides on the surface, wherein the surface is a metallic surface;

Production of EPS;

Bacterial promotion of the degradation of petroleum products in thesystem due to the release of the enzyme aryl hydrocarbon hydroxylase(AHH), wherein the surface is a metallic surface;

Production of sulphuric acid; and

Oxidation of sulphur.

Examples Applicable to Mic or Bifouling Control

There are specific example applications envisioned by the presentinvention to reduce the corrosion and/or biofouling associated withbacteria. The applications described below are not intended to limit theconcept of the present invention, and are merely illustrative of how theinvention may be used to control bacterially induced corrosion or toreduce environmental pollution.

Acid Mine Drainage:

In acid mine drainage, bacterial growth can increase acidity in theenvironment. A reaction scheme exists for the creation of acid and,therefore, potential environmental damage. The problem of acid minedrainage is recognised throughout the world as a severe environmentalproblem. The origin of acid mine drainage is the weathering andoxidation of pyretic and other sulphide containing minerals. Minedrainage is formed when pyrite, an iron sulfide, is exposed and reactswith air and water to form sulphuric acid and dissolved iron. Some orall of this iron can precipitate to form the red, orange, or yellowsediments in the bottom of streams containing mine drainage. The acidrun-off further dissolves heavy metals such as copper, lead, mercuryinto ground or surface water. The rate and degree by which acid-minedrainage proceeds can be increased by the action of certain bacteria.

In an example, the system is therefore a mine or comprised by a mine.The fluid is mine drainage fluid and the method reduces sulphuric acidcaused by mine drainage. In an example, the surface is in contact withmine drainage fluid.

In an example, the first host cells are Acidithiobacillus ferrooxidans,Acidithiobacillus thiooxidans, Acidithiobacillus denitrificans,Leptospirillum ferrooxidans or Sulfobacillus thermosulfidooxidans cellsor a mixture of two or more of these.

Hydraulic Fracturing: Hydraulic fracturing is a method to fracture rockformations to facilitate the extraction of gas and other hydrocarbons.Essentially, once a gas bearing formation is identified, wells are boredinto the earth in both vertical and horizontal directions to access thegas. The wells are then used to fracture the shale using high pressurewater, sand and a plethora of chemicals to maintain the fractures andfissures from being closed by the intense pressure of the overburdenonce the hydrofracturing is completed. Millions of gallons of water areused to frac a well. Between 30% and 70% of the frac fluid returns tothe surface as “flowback”. Flowback contains any matter that isdissolved in the frac water, including salt. What is dissolved dependson the location. The flowback is held in plastic lined pits at the wellsite until it is trucked and treated prior to disposal. At some point intime the high flow and relatively low salinity water converts to a lowerflow, but much higher salinity “produced water” to distinguish it from“flowback” water.

In either case the problem of microbially induced corrosion (MIC)exists. Of particular interest are the SRB. In an example, therefore,the system is hydraulic fracturing system and the fluid is a hydraulicfracturing liquid (eg, flowback water or produced water) or the surfaceto be treated is in contact with such a liquid. The method, for example,reduces SRB viability (eg, kills SRB and/or reduces SRB proliferation inthe liquid) and the first host cells are SRB.

For example, the first host cells are Acidithiobacillus bacteria,Acidithiobacillus thiooxidans, Ferrobacillus ferrooxidans, Thiobacillusthiooxidans, Thiobacillus thioparus, Thiobacillus concretivorus,Desulphovibrio (eg, salixigens, vulgaris, desulphuricans or africanus)or Desulphotomaculum (eg, orientis or nigrificans) cells, or a mixtureof two or more of these species.

Cooling Equipment (Eg, Cooling Towers):

The presence of bacteria in cooling equipment, such as cooling towers,can adversely affect the functioning of the cooling in several ways. Forexample, SRB support the creation of acid conditions on the walls ofcooling towers, heat exchangers, etc., which leads to corrosion andpotential shutdown of the cooling system while repairs are made.Additionally, biofilms on the walls of, for example, the heatexchangers, reduce the heat transfer coefficient of the heat exchangers,resulting in decreased operational efficiency of the cooling system.

Additionally, the corrosion of iron-containing components can beespecially detrimental. Oxidation of iron to iron(II) and reduction ofsulphate to sulphide ion with resulting precipitation of iron sulphideand generation of corrosive hydrogen ions in situ may take place via theSRB. The corrosion of iron by sulphate reducing bacteria is rapid and,unlike ordinary rusting, it is not self-limiting. Tubercles produced byDesulphovibrio consist of an outer shell of red ferric oxide mixed withblack magnetic iron oxide, containing a soft, black center of ferroussulphide.

In an example, therefore, the system is a cooling system and the fluidis a fluid (eg, water or an aqueous liquid) comprised by the system orthe surface to be treated is a surface of cooling equipment in contactwith such a fluid. In an example, the first host cells are SRB (eg, anySRB disclosed herein). In an example, the surface is an iron-containingsurface.

In an example, the first host cells are Legionella cells. Such speciesare detrimental to human health and propagated in water cooling,heating, processing or storage equipment. In an example, therefore, thesystem is such an equipment.

Pipeline Corrosion:

Hydrocarbon and petrochemical pipelines often include sufficientmoisture to permit bacterial growth, resulting in MIC eg, caused by SRB.The MIC is often caused by biofilms of aerobic bacteria which protectSRB which is anaerobic and in direct contact with the pipeline's innersurface. This creates acid conditions and other metal-corrodingconditions, which will result in localised corrosion and eventualfailure of the pipe.

In an example, the system comprises an equipment surface (eg, pipelineor drilling equipment) comprising a surface in contact with the firsthost cells (eg, SRB). For example, the system is a crude oil,hydrocarbon, petrochemical (eg, diesel or petroleum), gas or waterrecovery, processing, storage or transportation system. For example, thepipeline is a petrochemicals pipeline. For example, the pipeline iscomprised by an oil or gas rig. For example, the pipeline surface is incontact with sea water. For example, the pipeline surface is in contactwith a petrochemical fluid, crude oil or natural gas.

Wastewater Treatment:

Wastewater treatment involves adding activated sludge downstream of awastewater treatment plant in order to remove organic pollutants. Thus,after water is treated in a waste treatment facility, many organicpollutants are present which can be “digested” by bacteria. Thus, theactivated sludge is added to the treated water in a tank/container totreat the effluent from the wastewater treatment facility.

However, sometimes bacteria in the tank/container (whether originatingfrom the activated sludge, the wastewater itself, or the surroundingenvironment), will dominate and grow very rapidly. Such rapid growth canresult in a filamentous-shaped bacterial growth. Filaments can form upto 20-30% of the bacterial population in the tank or container, and theyfloat. This filamentous growth results in what is known as bulkingsludge. The present invention can be utilised for bulking sludgecontrol, which is an important Aspect in wastewater treatment.

Thus, in one example, the system is a water treatment system and thesurface is a surface of a container of the system, wherein the surfaceis in contact with water and the first host cells; or the fluid to betreated comprises said water and cells. In an example, the methodcontrols bacterial growth in sludge of a wastewater system.

Shipping & Transportation:

Ships and boats can experience MIC on their outer surfaces (eg, hulls)in contact with sea water or waterways (eg, rivers, lakes, ponds orfresh water). Inner hull surfaces can also be subject to MIC since theyare typically in contact with moisture or liquids that can harbourMIC-mediating microbes such as bacteria, for example in contact withballast water. For example, sea water is often carried in the hulls ofships (such as oil tankers) to provide stability at sea; such sea waterharbours bacteria that can mediate SRB. Other transportation vehicles,such as motor-driven vehicles (cars, trucks, vans or lorries), trains,spacecraft and aircraft can also be susceptible.

Thus, in one example, the system is a transportation vehicle (eg, fortransporting goods and/or people or livestock, eg, a cars, truck, van orlorry, train, spacecraft or aircraft). For example, the vehicle is aship or boat, eg, an oil, gas or petrochemicals sea vessel (eg, an oiltanker). In an example, the surface to be treated is in contact with seawater. In an example, the surface is an outer surface of a ship or boathull. In an example, the surface is an inner surface of a ship or boathull.

Bacterial Persistence or Growth (Biofouling) in Ballast Water:

A specific application of the invention is the treatment of marinevehicle (eg, ship or boat) ballast water to reduce undesirable bacteria,such as Vibrio cholerae, E coli and/or Enterococci sp.

Shipping moves over 90% of the world's commodities and is responsiblefor the global transfer of approximately 2-3 billion tons of ballastwater, which is routinely carried by ships to maintain their stability.A similar volume of ballast water may also be transferred domesticallywithin countries and regions each year (GloBallast Partnerships,www.globallast.imo.org). Ballast water has been recognized as the mainsource of invasive marine organisms that threaten naturally evolvedbiodiversity, the consequences of which are increasingly being realized(Anil et al. 2002). The unintentional introduction of disease-causingpathogenic bacteria, which are transported from the place of origin orformed during transportation, can have direct impact on society andhuman health. Ship ballast tanks hold different non-indigenousvertebrates, invertebrates, plants, microscopic algae, bacteria, etc.(Williams et al. 1988; Carlton and Geller 1993; Smith et al. 1996; Ruizet al. 2000; Drake et al. 2002, 2005, 2007; Mimura et al. 2005).Microorganisms, such as bacteria are introduced into alien environmentsin larger numbers than other organism owing to their high naturalabundance, capability to form resting stages, and capability towithstand a wide range of environmental conditions. Although all theorganisms taken onboard into ballast tanks may not survive, bacteria andmicro-algae are well capable of surviving prolonged periods ofunfavorable conditions by forming cysts, spores, or other physiologicalresting stages (Roszak et al. 1983; Hallegraeff and Bolch 1992; Anil etal. 2002; Carney et al. 2011). Once released these microorganisms arewell suited to be invasive owing to their small size which facilitatestheir passive dispersal and simpler requirements for survival thanmetazoans (Deming 1997). The concentration of cells of Vibrio species inballast samples examined from ships in Singapore Harbour were in therange of 1.1-3.9×10⁴ ml⁻¹ (Joachimsthal et al. 2004). The unintentionalintroduction of disease-causing pathogenic bacteria can have directsocietal impacts, including effects on human health. In an earlier studyit was found that most of the pathogens introduced to Chesapeake Bayoriginated from bacteria associated with plankton rather than the watercolumn itself (Ruiz et al. 2000). Thus, ballast water microorganismssuch as bacteria and archaea are of major concern in ballast watertreatment/management programs.

The International Maritime Organization (IMO) has developed a conventionaimed at preventing these harmful effects, adopting the InternationalConvention for the Control and Management of Ships' Ballast Water andSediments (the Ballast Water Management Convention) in 2004. In the US,the United States Coast Guard's Final Rule on Ballast Water Managemententered into force in June 2012, applying to ballast water discharge inUS waters.

Ballast-water exchange at sea is not considered an ideal method ofballast-water management, and considerable efforts are being made todevelop treatment methods. These methods must be in accordance withStandard D-2 of the IMO's Ballast Water Management Convention. StandardD2 specifies that treated and discharged ballast water must have:

fewer than ten viable organisms greater than or equal to 50 micrometersin minimum dimension per cubic metre

fewer than ten viable organisms less than 50 micrometres in minimumdimension and greater than or equal to 10 micrometers in minimumdimension per millilitre.

In addition, Standard D2 specifies that the discharge of the indicatormicrobes shall not exceed specified concentrations as follows:

toxicogenic Vibrio cholerae (O1 and O139) with less than onecolony-forming unit (cfu) per 100 millilitres or less than 1 cfu per 1gram (wet weight) zooplankton samples

Escherichia coli less than 250 cfu per 100 millilitres

intestinal Enterococci less than 100 cfu per 100 millilitres.

These are the indicator microbes, as a human health standard, but theyare not limited to these types. Indeed, it has been suggested that infact, in some cases the ballast water treatment used may make thingsworse. By removing small organisms that eat bacteria, some treatmentsystems have turned ballast tanks into bacteria incubators, so that thetreated discharges consistently contained higher concentrations ofbacteria, in some trials, thousands of times higher, than dischargesthat were left untreated. The increased bacteria may include humanpathogens.

In an example of the invention (eg, according to Aspect 70 or an Aspectdependent from Aspect 70), therefore, the system is a ship or boat ormarine vehicle (eg, a ship or boat, eg, an oil tanker in a harbour, dockor at sea). In an example, the fluid comprising the first host cells isballast water of ship or boat a marine vehicle (eg, ship or boat ballastwater, eg, oil tanker ballast water). In an example, the system is a seacontainer or a platform or rig (eg, oil or gas rig), eg at sea. In anexample, the fluid is ballast water of such a container, platform orrig.

In an embodiment, the detrimental bacteria (first host cells accordingto the invention, eg, according to Aspect 70 or an Aspect dependent fromAspect 70) are of a species selected from the group consisting of Vibriocholerae; Vibrio rumoiensis; Vibrio sp.; E coli; Enterococcus sp.;Pseudomonas synxantha; Pseudomonas stutzeri; Vibrio lentus;Pseudoalteromonas marina Pseudoalteromonas tetraodonis;Pseudoalteromonas sp.; Pseudomonas putida; Pseudomonas oleovorans;Vibrio splendidus; Vibrio cyclitrophicus; Enterococcus hirae;Enterococcus faecium Vibrio rotiferianus; Pseudoalteromonas undina;Serratia plymuthica; Pseudomonas fulva; Pseudomonas tolaasii;Pseudomonas stutzeri; Pseudomonas stutzeri; Vibrio tubiashii; Halomonasvenusta; Idiomarina loihiensis; Vibrio cyclitrophicus; Vibrio tubiashii;Serratia plymuthica; Pseudoalteromonas sp.; Pseudoalteromonas atlantica;Pseudomonas synxantha; Pseudomonas stutzeri; Pseudoalteromonascarrageenovora; Tenacibaculum sp.; Bacillus mycoides; Vibrio natriegens;Bacillus baekryungensis; Enterococcus hirae; Lactobacillus pentosus;Pseudoalteromonas carrageenovora; and Pseudomonas aeruginosa.

In an example, the first host cells are aerobic heterotrophic bacteria.In an example, the first host cells are Vibrio cholerae cells (eg,strain O1 and/or O139). In an example, the first host cells are E colicells. In an example, the first host cells are Enterococcus sp. cells.

“Characterization of Bacteria in Ballast Water Using MALDI-TOF MassSpectrometry”, Kaveh E et al, PLoS One. 2012; 7(6): e38515; Publishedonline 2012 Jun. 7. doi: 10.1371/journal.pone.0038515 (incorporatedherein by reference) discloses a suitable rapid and cost-effectivemethod for monitoring bacteria in ballast water.

A specific example of the invention is as follows:—

A method of controlling bacterial biofouling in ballast water of a shipor boat, wherein the water comprises a population of first host cells ofa first microbial species (such as Cholera, E coli or Enterococci sp)that mediates said biofouling, the method comprising

(i) contacting the population with a plurality of vectors that arecapable of transforming or transducing the cells, each vector comprisinga CRISPR array whereby CRISPR arrays are introduced into the host cells,wherein

(a) each CRISPR array comprises one or more sequences for expression ofa crRNA and a promoter for transcription of the sequence(s) in a hostcell; and

(b) each crRNA is capable of hybridising to a target sequence of a hostcell to guide Cas (eg, a Cas nuclease) in the host cell to modify thetarget sequence (eg, to cut the target sequence); the target sequencebeing a gene sequence for mediating host cell viability; and

(ii) allowing expression of said cRNAs in the presence of Cas in hostcells, thereby modifying target sequences in host cells, resulting inreduction of host cell viability and control of said biofouling.

In an example, step (i) comprises mixing the ballast water with thevectors, eg, in the hull of a ship or boat.

In an example, the ship or boat is a marine vehicle and the water is seawater. Instead of a ship or boat, in an alternative the ballast water iscomprised by a container or a drilling platform at sea, eg, an oilplatform or oil rig.

The invention also comprises a method of discharging ballast water froma ship or boat, wherein the discharged ballast water comprises watertreated by the method of the specific example above. In an example, thewater is discharged into a body of water, eg, a sea, ocean or waterway(eg, a river, canal, lake or reservoir).

The invention also comprises ship or boat ballast water comprisingCRISPR arrays, wherein the ballast water is obtained or obtainable bythe specific example above. The invention also comprises a sea containerballast water comprising CRISPR arrays, wherein the ballast water isobtained or obtainable by the specific example above. The invention alsocomprises ballast water of a platform or rig (eg, oil or gas rig) atsea, the water comprising CRISPR arrays, wherein the ballast water isobtained or obtainable by the specific example above. The arrays are asrecited in (a) and (b) of the specific example.

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The invention also provides vectors and CRISPR arrays as follows.

85. A vector comprising a CRISPR array for introduction into a bacterialhost cell, wherein the bacterium is capable of water-borne transmission,wherein

(a) the CRISPR array comprises a sequence for expression of a crRNA anda promoter for transcription of the sequence in a said host cell;(b) the crRNA is capable of hybridising to a host cell target sequenceto guide a Cas (eg, a Cas nuclease) in the host cell to modify thetarget sequence (eg, to cut the target sequence); the target sequencebeing a nucleotide sequence (eg, a gene or regulatory sequence) formediating host cell viability;(c) wherein the sequence of (a) comprises a sequence R1-S1-R1′ forexpression and production of the crRNA, wherein R1 is a first CRISPRrepeat, R1′ is a second CRISPR repeat, and R1 or R1′ is optional; and S1is a first CRISPR spacer that comprises or consists of a nucleotidesequence that is 80% or more identical (eg, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100% identical) to the host cell target sequence.

By “water-borne transmission” is meant that cells of said bacterium arecapable of being spread in water or an aqueous liquid between differentorganisms, within an organism, between different environments, orbetween an organism and an environment. Examples are Vibro cholera,Enterococcus spp and E coli. Vibrio cholerae is a gram negativecomma-shaped bacterium with a polar flagellum. It belongs to the classof the Gamma Proteobacteria. There are two major biotypes of V cholerae,classical and El Tor, and numerous serogroups. V cholerae is theetiological agent of cholera, a severe bacterial infection of the smallintestine, and a major cause of death in developing countries. Thepathogenicity genes of V cholerae are interesting targets to detect andto study V cholerae infections. Most of these genes are located in twopathogenicity islands, named TCP (Toxin-Coregulated Pilus) and CTX(Cholera ToXins), organized as prophages 1,2. TCP contains a cluster ofgenes involved in host adhesion via pili, while CTX genes are involvedin the synthesis of the cholera toxin 3.

In an embodiment, the vector is an isolated vector (ie, a vector not ina said host cell). In an example, the vector is an engineered orsynthetic vector (ie, a non-naturally occurring vector).

In an example, the array is an ICP1 array, ie, an array of an ICP1 Vcholerae phage, eg, wherein the phage is ICP1_2003_A, ICP1_2004_A,ICP1_2005_A, ICP1_2006_E or ICP1_20011_A. In an example the array is aCR1 or CR2 ICP1 phage array, eg, an engineered or non-naturallyoccurring derivative of such an array.

In an example, the CRISPR array and Cas are type 1-E or type 1-F, eg,subtype system 17.

In an example, the CRISPR array comprises a plurality of sequences, eachfor expression of a respective crRNA and a associated with a promoterfor transcription of the sequence in a said host cell.

In an example, the vector or each vector comprises a plurality (eg, 2,3, 4, 5, 6, 7, 8, 9, 10 or more) of said CRISPR arrays.

In an example, the vector comprises a nucleotide sequence encoding saidCas.

In another example, the vector is devoid of such a sequence. Forexample, in this case, the array(s) are operable with one or more Casproduced by the host cell.

86. The vector of Aspect 85, wherein the cell is a Vibrio cholerae,Enterococcus or E coli cell.

87. The vector of Aspect 85 or 86, wherein the vector is devoid of anucleotide sequence that is capable of expressing said Cas.

In an example, therefore the vector does not encode a Cas nuclease. Inan alternative the vector encodes a said Cas.

88. The vector of Aspect 85, 86 or 87, wherein the target sequence is aprotospacer sequence of 17-45 contiguous nucleotides, eg, 18, 19, 20 or21 contiguous nucleotides.

In an example, each spacer (S1) is a nucleotide sequence of 17-45contiguous nucleotides, eg, 18, 19, 20, 21, 30, 31, 32 or 33 contiguousnucleotides. The protospacer sequence for V cholerae PLE is, forexample, 32 contiguous nucleotides and a vector targeting this can, forexample, have a spacer sequence of 32 contiguous nucleotides that is100% or at least 80, 90 or 95% identical to the 32 nucleotide PLEsequence. Where the vector comprises a plurality of spacers, the spacerscan be a mixture of different spacers, or can be identical spacers. Forexample, the array comprises a plurality of spacers, wherein a sub-setof spacers are identical. The identical spacers can be homologous toprotospacer sequence of a gene encoding a pathogenicity factor of thehost cell, for example. Using multiple spacers may be advantageous ifthe host cuts one or more of the spacers once the vector is inside thecell—uncut spacers are still able to form crRNAs and home to targetsequences. Using a mixture of different spacers in the vector or in anarray is advantageous to minimise the risk of adaptation of the host tothe invading vector, thereby minimising resistance.

89. The vector of any one of Aspects 85 to 88, wherein the targetsequence is a virulence, resistance or essential gene sequence. In anexample, the target sequence is a sequence of a PICI-like element (PLE),eg, a V cholerae PLE. Eg, PLE1.

90. The vector of any one of Aspects 85 to 89, wherein the targetsequence is a pathogenicity island sequence, optionally wherein the hostcell is a Vibrio cholera cell and the target sequence is a TCP, CTX orVPI sequence. In an example (eg, wherein the host is Vibrio)pathogenicity island is TCP (Toxin-Coregulated Pilus) or CTX (CholeraToXins). The Vibrio pathogenicity island (VPI) contains genes primarilyinvolved in the production of toxin coregulated pilus (TCP). It is alarge genetic element (about 40 kb) flanked by two repetitive regions(att-like sites), resembling a phage genome in structure. The VPIcontains two gene clusters, the TCP cluster, and the ACF cluster, alongwith several other genes. The acfcluster is composed of four genes:acfABCD. The tcp cluster is composed of 15 genes: tcpABCDEFHIJPQRST andregulatory gene toxT.

91. The vector of any one of Aspects 85 to 90, wherein the host cell isVibrio cholera and the target sequence is a CTXφ gene sequence. Thegenes for cholera toxin are carried by CTXphi (CTXφ), a temperatebacteriophage inserted into the V. cholerae genome. CTXφ can transmitcholera toxin genes from one V. cholerae strain to another, one form ofhorizontal gene transfer. The genes for toxin coregulated pilus arecoded by the VPI pathogenicity island (VPI).

92. The vector of any one of Aspects 85 to 90, wherein the host cell isVibrio cholera and the target sequence is a ctxB, tcpA, ctxA, tcpB,wbet, hlyA, hapR, rstR, mshA or tcpP sequence.

93. The vector of any one of Aspects 85 to 92, wherein the targetsequence is 17-45 contiguous nucleotides (eg, 18, 19, 20 or 21contiguous nucleotides) and at least 80% (eg, 85, 90, 91, 92, 93, 94,95, 96, 97, 98, 99 or 100% identical) identical to a sequence of a phageinducible chromosomal island (PICI) of a Gram-positive bacterium, eg, aStaphylococcus aureus pathogenicity island (SaPI). Examples of ICP1phage (aka ICP1-related phage) spacer sequences are provided below.These spacer sequences are homologous to target sequences (protospacersequences) in V cholera.

94. The vector of any one of Aspects 85 to 93, wherein the host cell isVibrio cholera and the crRNA is capable of hybridising to a targetsequence within 5, 4 or 3 nucleotides of a protospacer adjacent motif(PAM) in a Vibrio cholerae cell, wherein the PAM is GA.

95. The vector of any one of Aspects 85 to 94, wherein the host cell isVibrio cholera and the cell is an El Tor, O1 or O139 Vibrio choleraecell. In an example, the V. cholerae is serotype O1 El Tor N16961; ElTor biotype18; or El Tor strain MJ-1236. In an example, the host cell isa E. coli O157:H7 cell.

96. The vector of any one of Aspects 85 to 95, wherein the vector is abacteriophage that is capable of infecting a said host cell. In anexample, the host cell is E coli, and the phage is a lambda or T4 phage.In an example, the host cell is an Enterococcus cell and the phage is aEnterococcus phage IME-EF1, phiEF24C, φEf1 or EFDG1 (see Appl EnvironMicrobiol. 2015 April; 81(8):2696-705. doi: 10.1128/AEM.00096-15. Epub2015 Feb. 6, “Targeting Enterococcus faecalis biofilms with phagetherapy”, Khalifa L et al).

97. The vector of Aspect 96, wherein the host cell is Vibrio cholera andthe vector is a bacteriophage capable of infecting a Vibrio choleraecell.

98. The vector of Aspect 97, wherein the bacteriophage is selected fromCTXφ, an ICPI phage and a myovirus, eg, wherein the phage isICP1_2003_A, ICP1_2004_A, ICP1_2005_A, ICP1_2006_E or ICP1_20011_A,optionally an engineered and non-naturally occurring phage.

99. The vector of any one of Aspects 85 to 98, wherein the vector is orcomprises an ICE, eg, a transposon. The ICE can comprise any of thefeatures of an ICE described herein.

100. The vector of Aspect 99, wherein the transposon is a conjugativetransposon capable of transfer from a first to a second said host cell.

101. The vector of Aspect 99 or 100, wherein the transposon leaves acopy of the CRISPR array in the first cell.

102. The vector of any one of Aspects 85 to 101, wherein the or eacharray is comprised by a respective mobile genetic element (MGE), whereinthe MGE comprises an origin of transfer (oriT) operable in the hostcell. The MGE can be according to any MGE described herein.

103. The vector of any one of Aspects 85 to 102, wherein the vector isan engineered vector.

104. A water or food treatment composition comprising a plurality ofvectors according to any one of Aspects 85 to 103.

In an example, the water is ballast water, sea water, brackish water,fresh water, drinking water, waterway water (eg, estuary water) orindustrial water. In an example, the water is water in human GI tractfluid.

In an example, the host cell is comprised by shellfish, fish, rice orgrains. In an example, the composition is for treating food and the hostcell is a E. coli O157:H7 cell. In an example, the target sequence is asequence encoding a Shiga toxin in an E coli (eg, O157:H7) host cell. Inan alternative to the water-borne species described so far, the hostcell is a Salmonella or Listeria cell.

105. A medicament for treatment or prevention of Vibrio choleraeinfection in a human, the medicament comprising a plurality of vectorsaccording to any one of Aspects 85 to 103. In an alternative, theinvention provides a medicament for treatment or prevention of E coliinfection in a human, the medicament comprising a plurality of vectorsof the invention. In an alternative, the invention provides a medicamentfor treatment or prevention of Enterococcus infection in a human, themedicament comprising a plurality of vectors of the invention.

106. The composition or medicament of Aspect 104 or 105, furthercomprising an anti-host cell antibiotic or an anti-host cell biocide.

Example 5 below is an example relating to cholera.

Any of the general features (see below) also may apply to the presentconfiguration (sixth configuration). Any configuration below iscombinable with the present configuration, eg, to provide combinationsof features for inclusion in one or more claims herein.

Regulating Cas Activity

These aspects of the invention are useful for regulating Cas activity,eg, in a cell or in vitro. The invention involves targeting aCas-encoding gene to restrict Cas activity, which is advantageous fortemporal regulation of Cas. The invention may also be useful in settingswhere increased stringency of Cas activity is desirable, eg, to reducethe chances for off-target Cas cutting in when modifying the genome of acell. Applications are, for example, in modifying human, animal or plantcells where off-target effects should be minimised or avoided, eg, forgene therapy or gene targeting of the cell or a tissue or an organismcomprising the cell. For example, very high stringency is required whenusing Cas modification to make desired changes in a human cell (eg, iPScell) that is to be administered to a patient for gene therapy or fortreating or preventing a disease or condition in the human. Thedisclosure provides these applications as part of the methods andproducts of the invention.

The invention thus provides the following clauses:—

-   1. A method of modifying an expressible gene encoding a first Cas,    the method comprising    -   (a) combining a guide RNA (gRNA1) with the Cas gene in the        presence of first Cas that is expressed from said gene; and    -   (b) allowing gRNA1 to hybridise to a sequence of said Cas gene        (eg, a promoter or a first Cas-encoding DNA sequence thereof)        and to guide first Cas to the gene, whereby the Cas modifies the        Cas gene.

In an example, the method is a cell-free method (eg, recombineeringmethod) in vitro. In another example, the method is carried out in acell, eg, wherein the gene is cut by Cas that it encodes itself (ie,endogenous Cas is used to cut the gene).

-   2. The method of clause 1, wherein the Cas is a nuclease and the Cas    gene is cut.-   3. The method of clause 1 or 2, wherein the Cas gene is mutated,    down-regulated or inactivated.-   4. The method of any one of clauses 1 to 3, wherein the first Cas is    a Cas9.-   5. The method of any one of clauses 1 to 4, wherein gRNA1 is a    single guide RNA.-   6. The method of any one of clauses 1 to 5, wherein the method is    carried out in a host cell.-   7. The method of clause 6, wherein the cell is a prokaryotic cell,    eg, a bacterial or archaeal cell (eg, an E coli cell).-   8. The method of clause 6, wherein the method is a recombineering    method.-   9. The method of clause 6, 7 or 8, wherein the cell is of a human or    non-human animal pathogen species or strain (eg, S aureus).-   10. The method of any one of clauses 6 to 9, wherein the cell is a    cell of a human microbiome species, eg, a human gut microbiome    species.-   11. The method of any one of clauses 6 to 10, wherein the Cas gene    is comprised by a host CRISPR/Cas system.

Optionally an exogenous first Cas-encoding sequence is not used in themethod, for example when the host cell comprises a wild-type endogenousCas nuclease that is cognage to gRNA1.

-   12. The method of clause 6, wherein the cell is a eukaryotic cell    (eg, a human, non-human animal, yeast or plant cell).-   13. The method of clause 12, wherein the method is carried out in a    non-human embryo; non-human zygote; non-human germ cell; or a human    or animal (eg, wherein the method is a cosmetic method); optionally    wherein the method is not a method for treatment of the human or    animal body by surgery or therapy or diagnosis.-   14. The method of any one of clauses 6 to 3, wherein the Cas gene is    comprised by a nucleic acid that is introduced into the cell in step    (a).-   15. The method of any one of clauses 6 to 4 for reducing the    development of host cell resistance to transformation by a nucleic    acid vector or maintenance of a nucleic acid vector in the host    cell.-   16. The method any one of clauses 1 to 15, or a nucleic acid or cell    product thereof for human or animal medical therapy, prophylaxis or    diagnosis (eg, for gene therapy of a human or animal, human or    animal cell when the method is carried out in a human or animal    cell; or for treating or preventing a bacterial infection in a human    or animal when the method is carried out in a bacterial cell).-   17. The method any one of clauses 1 to 6, wherein the method is    carried out in vitro.-   18. The method any one of clauses 1 to 16, wherein the method is    carried out in vivo, optionally not in a human embryo and optionally    wherein the method is not a method for treatment of the human or    animal body by surgery or therapy or diagnosis.-   19. The method any one of clauses 1 to 18, wherein gRNA1 is produced    by transcription from a first nucleic acid that is combined with the    Cas gene in step (a).-   20. The method of clause 19, wherein the method is carried out in a    cell and the first nucleic acid encoding gRNA1 is introduced into    the cell in step (a); or a first nucleic acid encoding a crRNA is    introduced into the cell in step (a) wherein the crRNA forms gRNA1    with a tracrRNA in the cell.-   21. The method of clause 19 or 20, wherein the Cas gene is combined    with a target nucleic acid comprising a target site (CS-t) to be    modified by first Cas, and wherein    -   I. the Cas gene comprises a first protospacer (PS1) adjacent a        PAM (P1) that is cognate to the first Cas, wherein PS1 is        modified (eg, cut) at a first site (CS1) by first Cas;    -   II. gRNA1 comprises a sequence that is complementary to PS1 for        guiding first Cas wherein PS1 is modified at CS1 in step (b);    -   III. the target nucleic acid comprises a protospacer sequence        (PS-t) adjacent a PAM (P-t), wherein P-t is cognate to the first        Cas;    -   IV. before or during step (b) the method comprises combining a        guide RNA (gRNA-t) with the target nucleic acid and first Cas        expressed from said gene, wherein gRNA-t hybridises to PS-t and        guides first Cas to modify CS-t; and    -   V. the method optionally comprises isolating or sequencing the        modified target nucleic acid.-   22. The method of any clause 21, wherein gRNA-t is produced by    transcription from a nucleic acid (eg, said first nucleic acid) that    is combined with the Cas in step IV.-   23. The method of clause 22, wherein the method is carried out in a    cell and the nucleic acid encodes a crRNA, wherein the crRNA forms    gRNA-t with a tracrRNA in the cell.-   24. The method of any one of clauses 21 to 23, wherein the    production of gRNA1 is commenced after the production of gRNA-t,    whereby PS-t is modified (eg, cut) in copies of the target nucleic    acid before PS1 is modified (eg, cut) to down-regulate or inactivate    first Cas expression.-   25. The method of any one of clauses 1 to 24, further comprising    combining the cut target nucleic acid with a further nucleic acid,    whereby homologous recombination between the nucleic acids takes    place and    -   (i) a nucleotide sequence of the target nucleic acid is deleted;    -   (ii) a nucleotide sequence of the further nucleic acid is        deleted;    -   (iii) a nucleotide sequence of the target nucleic acid is        inserted into the further nucleic acid; and/or    -   (iv) a nucleotide sequence of the further nucleic acid is        inserted into the target nucleic acid.-   26. The method of clause 25, wherein (i) takes place, thereby    inactivating a nucleotide sequence or regulatory element of the    target nucleic acid.-   27. The method of clause 25, wherein (i) takes place, thereby    activating a nucleotide sequence or regulatory element of the target    nucleic acid.-   28. The method of clause 25, 26 or 27, wherein (ii) takes place,    thereby inactivating a nucleotide sequence or regulatory element of    the further nucleic acid.-   29. The method of clause 25, 26 or 27, wherein (ii) takes place,    thereby activating a nucleotide sequence or regulatory element of    the further nucleic acid.-   30. The method of any one of clauses 25 to 29, wherein (iii) takes    place, optionally placing the inserted sequence in functional    relationship with a regulatory element of the further nucleic acid    and/or creating a new marker sequence.-   31. The method of any one of clauses 25 to 30, wherein (iv) takes    place, optionally placing the inserted sequence in functional    relationship with a regulatory element of the target nucleic acid    and/or creating a new marker sequence.-   32. The method of clause 30 or 31, further comprising detecting the    new marker sequence or an expression product thereof to determine    that homologous recombination has taken place.-   33. The method of any one of clauses 21 to 32, further comprising    isolating or sequencing the target nucleic acid product, the further    nucleic acid product and/or the first vector product.-   34. The method of any one of clauses 1 to 33, wherein the first    vector is as defined in any one of clauses 35 to 55.-   35. A first (eg, isolated) nucleic acid vector or combination of    vectors, eg, for use in the method of clause 1, wherein    -   (a) the first vector or a vector of said combination comprises        an expressible nucleotide sequence that encodes a guide RNA        (gRNA1, eg, a single gRNA) that is complementary to a        predetermined protospacer sequence (PS1) for guiding a first Cas        to modify PS1 at a first site (CS1), wherein PS1 is adjacent a        PAM (P1) that is cognate to the first Cas; or the expressible        sequence encodes a crRNA that forms gRNA1 with a tracrRNA; and    -   (b) PS1 and P1 are sequences of an expressible first        Cas-encoding gene and PS1 is capable of being modified at CS1 by        the first Cas.

Each vector herein in any configuration can be a linear or circular (eg,closed circular, optionally supercoiled) DNA carrying the specifiedsequence(s).

-   36. The vector or combination of clause 35, wherein the first Cas is    a nuclease, wherein CS1 is capable of being cut by the nuclease.-   37. The vector or combination of clause 35 or 36, wherein the first    Cas is a Cas9.-   38. The vector or combination of any one of clauses 35 to 37,    wherein gRNA1 is a single guide RNA.-   39. The vector or combination of any one of clauses 35 to 38,    wherein the nucleotide sequence is expressible in a prokaryotic cell    (eg, a bacterial or archaeal cell) for producing gRNA1.-   40. A recombineering kit comprising the vector or combination of    clause 39 (eg, wherein the cell is a recombineering-permissive E    coli cell).-   41. The vector or combination of any one of clauses 35 to 38,    wherein the nucleotide sequence is expressible in a eukaryotic cell    (eg, a human, animal, plant or yeast cell) for producing gRNA1.-   42. The vector or combination of any one of clauses 35 to 41,    wherein the first vector or a vector of said combination (eg, the    second vector) comprises an expressible nucleotide sequence that    encodes a guide RNA (gRNA-t, eg, a single gRNA) that is    complementary to a predetermined protospacer sequence (PS-t) of a    target nucleic acid for guiding first Cas to modify (eg, cut) PS-t;    or the expressible sequence encodes a crRNA that forms gRNA-t with a    tracrRNA; the target nucleic acid comprises PS-t adjacent a PAM    (P-t), wherein P-t is cognate to the first Cas for modifying PS-t.-   43. The vector or combination of clause 42, further in combination    with said target nucleic acid.-   44. The vector or combination of clause 43, wherein said target    nucleic acid is a chromosomal or episomal nucleic acid of a cell.-   45. The vector or combination of clause 44, wherein the cell is the    cell is of a human or non-human animal pathogen species or strain    (eg, S aureus).-   46. The vector or combination of clause 44 or 45, wherein the cell    is a cell of a human microbiome species, eg, a human gut microbiome    species.-   47. The vector or combination of any one of clauses 44 to 46,    wherein PS-t is comprised by an essential gene, virulence gene or    antibiotic resistance gene sequence of the cell (eg, a prokaryotic    cell).-   48. The vector or combination of clause 47, wherein the gene is    down-regulated or inactivated when first Cas modifies (eg, cuts)    PS-t.-   49. The vector or combination of clause 47, wherein the gene is    up-regulated or activated when first Cas modifies PS-t.-   50. The vector or combination of any one of clauses 35 to 49 in    combination with said gene encoding the first Cas (eg, comprised by    the first vector).-   51. The vector or combination of any one of clauses 35 to 50 when    inside a cell, wherein the cell comprises a CRISPR/Cas system    comprising said gene encoding the first Cas.-   52. The vector or combination of any one of clauses 35 to 51 for    treating, preventing or diagnosing a disease or condition in a human    or non-human animal, eg, for gene therapy of a human or animal,    human or animal cell when the method is carried out in a human or    animal cell; or for treating or preventing a bacterial infection in    a human or animal when the method is carried out in a bacterial    cell.-   53. A foodstuff, food ingredient or precursor ingredient, beverage,    water (eg, intended for human consumption), an industrial or    environmental substance (eg, oil, petroleum product, soil or a    waterway or reservoir; or equipment for recovering or processing    oil, petroleum product, soil, water, a foodstuff, foodstuff    ingredient or precursor, or a beverage or beverage ingredient of    precursor) comprising a first vector or combination according to any    one of clauses 35 to 52.-   54. An antibiotic (eg, anti-bacterial or anti-archaeal) composition    a first vector or combination according to any one of clauses 35 to    52.-   55. A medicament for treating or preventing a disease or condition    (eg, a bacterial infection or obesity) in a human or animal, the    medicament comprising a first vector or combination according to any    one of clauses 35 to 52.

In an example, the vector, combination, medicament or antibiotic iscomprised by a medical device or medical container (eg, a syringe,inhaler or IV bag).

Any of the general features (see below) also may apply to the presentconfiguration. Any configuration below is combinable with the presentconfiguration, eg, to provide combinations of features for inclusion inone or more claims herein.

Generally Applicable Features

The following features apply to any configuration (eg, in any of itsaspects, embodiments, concepts, paragraphs or examples) of theinvention:—

In an example, the target sequence is a chromosomal sequence, anendogenous host cell sequence, a wild-type host cell sequence, anon-viral chromosomal host cell sequence, not an exogenous sequenceand/or a non-phage sequence (ie, one more or all of these), eg, thesequence is a wild-type host chromosomal cell sequence such as anantibiotic resistance gene or essential gene sequence comprised by ahost cell chromosome. In an example, the sequence is a host cell plasmidsequence, eg, an antibiotic resistance gene sequence.

In an example, at least two target sequences are modified by Cas, forexample an antibiotic resistance gene and an essential gene. Multipletargeting in this way may be useful to reduce evolution of escape mutanthost cells.

In an example, the Cas is a wild-type endogenous host cell Cas nucleaseand/or each host cell is a wild-type host cell. Thus, in an embodimentthe invention uses host cells without the need to de-repress endogenousCas first to provide relevant Cas activity. In an example, each hostcell has constitutive Cas nuclease activity, eg, constitutive wild-typeCas nuclease activity. In an example, the host cell is a bacterial cell;in an other example the host cell is an archael cell. Use of anendogenous Cas is advantageous as this enables space to be freed invectors encoding HM- or PM-cRNA or gRNA. For example, Type II Cas9nucleotide sequence is large and the use of endogenous Cas of the hostcell instead is advantageous in that instance when a Type II CRISPR/Cassystem is used for host cell modification in the present invention. Themost commonly employed Cas9, measuring in at 4.2 kilobases (kb), comesfrom S pyogenes. While it is an efficient nuclease, the molecule'slength pushes the limit of how much genetic material a vector canaccommodate, creating a barrier to using CRISPR in the tissues of livinganimals and other settings described herein (see F. A. Ran et al., “Invivo genome editing using Staphylococcus aureus Cas9,” Nature, doi:10.1038/nature 14299, 2015). Thus, in an embodiment, the vector of theinvention is a AAV vector or has an exogenous DNA insertion capacity nomore than an AAV vector, and the Cas is an endogenous Cas of the hostcell, wherein the cell is a bacterial or archaeal cell.

S thermophilus Cas9 (UniProtKB-G3ECR1 (CAS9_STRTR)) nucleotide sequencehas a size of 1.4 kb.

In an embodiment, therefore, the invention provides

A nucleic acid vector comprising more than 1.4 kb or more than 4.2 kb ofexogenous DNA sequence encoding components of a CRISPR/Cas system,wherein the sequence comprises an engineered array or engineeredsequence (optionally as described herein) for expressing one or more HM-or PM-crRNAs or gRNAs in host cells (any cell herein, eg, human, anialor bacterial or archael host cells), wherein the array or engineeredsequence does not comprise a nucleotide sequence encoding a Cas nucleasethat is cognate to the cRNA(s) or gRNA(s); optionally wherein at least2, 3 or 4 cRNAs or gRNAs are encoded by the exogenous DNA. In anembodiment, the host cell is a bacterial or archael cell that expressesa Cas nuclease that is cognate to the crRNAs or gRNAs. In anotherexample, such as for use with human or animal (eg, rodent, rat or mouse)cells the Cas nuclease is encoded by a different nucleic acid vector. Inan example, wherein the cell is a human or animal cell, the vector is anAAV or lentiviral vector. In an example, the invention comprises a hostcell comprising such a vector, wherein the host cell expresses said Cas.In an example, the host cell is a human or animal cell ex vivo.

The invention also provides

A nucleic acid vector comprising more than 1.4 kb or more than 4.2 kb ofexogenous DNA sequence, wherein the exogenous DNA encodes one or morecomponents of a CRISPR/Cas system and comprises an engineered array orsequence (eg, any such one described herein) for expressing one or moreHM-crRNAs or gRNAs in host cells, wherein the exogenous sequence isdevoid of a nucleotide sequence encoding a Cas nuclease that is cognateto the cRNA(s) or gRNA(s); optionally wherein at least 2 different cRNAsor gRNAs are encoded by the exogenous DNA. In an example, the inventioncomprises a host cell comprising such a vector, wherein the host cellexpresses said Cas. In an example, the cRNAs or gRNAs are capable ofhybridising in host cells to respective target protospacer sequences,wherein each protospacer sequence is comprised by an antibioticresistance or essential host gene. This is exemplified by the workedexamples herein where we show selective host cell growth inhibition byat least 10-fold in a mixed and non-mixed cell population. The mixturesimulates a combination of species and strains found in humanmicrobiota.

By “exogenous DNA sequence encoding components of a CRISPR/Cas system”is meant DNA that is inserted into a vector backbone, or such DNA in aprogeny of a vector into which said insertion has previously taken place(eg, using recombinant DNA technology, such as recombineering). In anexample, the exogenous DNA is 95, 90, 80, 85, 70 or 60% of the insertioncapacity of the vector.

In an example, the vector is a viral vector. Viral vectors have aparticularly limited capacity for exogenous DNA insertion, thus viruspackaging capacity needs to be considered. Room needs to be left forsequences encoding vital viral functions, such as for expressing coatproteins and polymerase. In an example, the vector is a phage vector oran AAV or lentiviral vector. Phage vectors are useful where the host isa bacterial cell.

The invention provides a combination product kit (eg, for treating orpreventing a disease or condition in a human or animal subject asdescribed herein), wherein the kit comprises an array, vector, system,cell, engineered cRNA or gRNA-encoding sequence or the cRNA or gRNA,which is in combination with an antibiotic (first antibiotic), whereinthe cRNA or gRNA is capable of hybridising to a protospacer sequencecomprised by a bacterial host cell antibiotic resistance gene whereinthe antibiotic is said first antibiotic. The antibiotic can be anyantibiotic disclosed herein. In an embodiment, the antibiotic iscombined in a formulation with the array, vector, system, cell,engineered cRNA or gRNA-encoding sequence or the cRNA or gRNA. In anexample, the kit comprises the antibiotic in a container separate from acontainer comprising the array, vector, system, cell, engineered cRNA orgRNA-encoding sequence or the cRNA or gRNA.

In an embodiment, unless otherwise specified the or each cell is abacterial cell, archaeal cell, algal cell, fungal cell, protozoan cell,invertebrate cell, vertebrate cell, fish cell, bird cell, mammal cell,companion animal cell, dog cell, cat cell, horse cell, mouse cell, ratcell, rabbit cell, eukaryotic cell, prokaryotic cell, human cell, animalcell, rodent cell, insect cell or plant cell. Additionally, in this casepreferably the cells are of the same phylum, order, family or genus.

By use of the term “engineered” it will be readily apparent to theskilled addressee that the array, sequence, vector, MGE or any otherconfiguration, concept, aspect, embodiment, paragraph or example etc ofthe invention is non-naturally occurring. For example, the MGE, vectoror array comprises one or more sequences or components not naturallyfound together with other sequences or components of the MGE, vector orarray. For example, the array is recombinant, artificial, synthetic orexogenous (ie, non-endogenous or not wild-type) to the or each hostcell.

In an example, the array or vector of the invention is isolated, forexample isolated from a host cell. In an example, the array or vector isnot in combination with a Cas endonuclease-encoding sequence that isnaturally found in a cell together with repeat sequences of the array.

In an example, the vector, MGE or array is not in combination with a Casendonuclease-encoding sequence when not in a host cell. In an example,the vector or MGE does not comprise a Cas endonuclease-encodingsequence.

In an example, the target modification or cutting is carried out by adsDNA Cas nuclease (eg, a Cas9, eg, a spCas9 or saCas9), whereby repairof the cut is by non-homologous end joining (NHEJ). This typicallyintroduces mutation (indels) at the repair site, which is useful forinactivation of the target site (eg, phage gene or regulatory element,such as an essential gene or regulatory element thereof). In anotherexample, the cutting is carried out by a ssDNA Cas nuclease (eg, a Cas9nuclease) that cuts in a single strand (but does not do double strandedDNA cuts). This is useful for favouring HDR repair of the cut, whichreduces the chances of indels. This may be useful where the target site(or gene or regulatory element comprising it) is desired, eg, where aHM- or PM-DNA is inserted at the target site for desired modification ofthe site. For example, in this case the modified gene produces a fusionprotein comprising HM-DNA-encoded amino acid fused to host DNA-encodedsequence, or PM-DNA-encoded amino acid sequence fused to phageDNA-encoded sequence. The invention also provides a sequence encoding afusion protein obtained or obtainable by such a method. In anotherexample, the HM- or PM-DNA comprises a regulatory element (eg apromoter, enhancer, repressor or inducible switch for regulating geneexpression), such that the fusion product comprises said DNA fused tohost or phage gene or regulatory element DNA, thereby producing a fusiongene. The invention also provides a fusion gene obtained or obtainableby such a method. In an embodiment, the invention provides a vector (eg,a virus, virion, phage, phagemid, prophage or plasmid) comprising such afusion gene, optionally wherein the vector is contained in a bacterialcell (eg, a prokaryotic, eukaryotic, bacterial, archaeal or yeast cell).In an example, the cell is in vitro.

In an example, the HM- or PM-DNA is vector DNA inside the cell. Forexample, the HM- or PM-DNA in the vector can be flanked by site-specificrecombination sites (eg, frt or lox sites) which are cut by the actionof a site-specific recombinase which is encoded by either a host cell orvector sequence. In another example, the vector comprises DNA that istranscribed into RNA copies of the HM- or PM-DNA and a reversetranscriptase (eg, encoded by the vector nucleic acid sequence) forproducing HM- or PM-DNA from the RNA. This is useful for producing manycopies of the desired HM- or PM-DNA to increase the chances of efficientand effective introduction at one or more of the target sites. Inanother embodiment, the HM- or PM-DNA is, or is encoded by, nucleic acidof a second vector (eg, a second phage or plasmid) that has transducedor transformed the host cell. For example, this could be a helper phage(which may also encode one or more coat proteins required for packagingof the first page vector). In another example, the DNA is provided invector DNA and flanked by arms, wherein the 5′ arm comprises a PAM thatis recognised by a Cas nuclease when the vector is contained in the hostcell and the 3′ arm is flanked immediately downstream (3′) by such aPAM, whereby in the host cell Cas cleavage liberates the HM- or PM-DNAwith its flanked arms that can be designed to be homologous to hostsequences flanking the cut in the host target sequence, whereby theHM-DNA is integrated into the host genome, or the PM-DNA is integratedinto the phage genome. In one aspect, the invention provides a nucleicacid comprising such a HM- or PM-DNA and arms or a vector (eg, a phageor packaged phage) comprising such a nucleic acid, optionally whereinthe vector is contained by a host cell. Optionally, the HM-DNA is incombination with a HM-array as herein defined. Optionally, the PM-DNA isin combination with a PM-array as herein defined.

A particular application of the invention is the alteration of theproportion of Bacteroidetes (eg, Bacteroides) bacteria in a mixedbacterial population ex- or in vivo. As discussed above, this may beuseful for environmental treatment such as treatment of waterways ordrinking water infected with undesired Bacteroidetes, or for favouringuseful commensal or symbiotic Bacteroidetes in humans or animals, eg,for producing bacterial cultures for administration to humans or animalsfor such purpose. In an example of the latter, the invention is usefulfor increasing the relative ratio of Bacteroidetes versus Firmicutes,which has been associated with lower of body mass and thus finds utilityin treating or preventing obesity for medical or cosmetic purposes.

Studies suggest that Bacteroides have a role in preventing infectionwith Clostridium difficile. The development of the immune response thatlimits entry and proliferation of potential pathogens is profoundlydependent upon B fragilis. Also, Paneth cell proteins may produceantibacterial peptides in response to stimulation by B thetaiotomicron,and these molecules may prevent pathogens from colonizing the gut. Inaddition, B thetaiotomicron can induce Paneth cells to produce abactericidal lectin, RegIII, which exerts its antimicrobial effect bybinding to the peptidoglycan of gram-positive organisms. Thus, the useof the invention in any of its configurations for increasing theproportion of Bacteroides (eg, thetaiotomicron and/or fragalis) in amixed population of gut bacteria is useful for limiting pathogenicbacterial colonisation of the population or a gut of a human ornon-human animal.

Hooper et al demonstrated that B thetaiotomicron can modify intestinalfucosylation in a complex interaction mediated by a fucose repressorgene and a signaling system. Using transcriptional analysis it wasdemonstrated that B thetaiotaomicron can modulate expression of avariety of host genes, including those involved in nutrient absorption,mucosal barrier fortification, and production of angiogenic factors.

In an embodiment, the mixed population consists of the first and secondbacteria (ie, and no further bacterial population).

In an example, the or each array is recombinant array in a vector and/oran isolated array in a vector. In an example, the array is contained ina host cell (eg, a microbial, bacterial or archaeal cell).

In an example, said Cas is an endogenous Cas nuclease (eg Cas9) of thehost cell. By harnessing the Cas of the host, this enables efficient useof host-type repeats in the array and possibility of using endogenouscrRNA too—freeing up capacity which is otherwise limited in vectors,such as viruses or phage (noting that the Cas gene sequence such as TypeII Cas9 is large).

In an example, the host CRISPR/Cas system is a Type I system. In anexample, the host CRISPR/Cas system is a Type II system. In an example,the host CRISPR/Cas system is a Type III system.

The cas guided by the HM-crRNA or gRNA of the invention is a hostendogenous Cas or a vector-encoded Cas compatible with the PAM in thetarget sequence.

Optionally, the host (or first and/or second bacteria) is a gramnegative bacterium (eg, a spirilla or vibrio). Optionally, the host (orfirst and/or second bacteria) is a gram positive bacterium. Optionally,the host (or first and/or second bacteria) is a mycoplasma, chlamydiae,spirochete or mycobacterium. Optionally, the host (or first and/orsecond bacteria) is a Streptococcus (eg, pyogenes or thermophilus) host.Optionally, the host (or first and/or second bacteria) is aStaphylococcus (eg, aureus, eg, MRSA) host. Optionally, the host (orfirst and/or second bacteria) is an E. coli (eg, O157: H7) host, eg,wherein the Cas is encoded by the vector or an endogenous host Casnuclease activity is de-repressed. Optionally, the host (or first and/orsecond bacteria) is a Pseudomonas (eg, aeruginosa) host. Optionally, thehost (or first and/or second bacteria) is a Vibro (eg, cholerae (eg,O139) or vulnificus) host. Optionally, the host (or first and/or secondbacteria) is a Neisseria (eg, gonnorrhoeae or meningitidis) host.Optionally, the host (or first and/or second bacteria) is a Bordetella(eg, pertussis) host. Optionally, the host (or first and/or secondbacteria) is a Haemophilus (eg, influenzae) host. Optionally, the host(or first and/or second bacteria) is a Shigella (eg, dysenteriae) host.Optionally, the host (or first and/or second bacteria) is a Brucella(eg, abortus) host. Optionally, the host (or first and/or secondbacteria) is a Francisella host. Optionally, the host (or first and/orsecond bacteria) is a Xanthomonas host. Optionally, the host (or firstand/or second bacteria) is a Agrobacterium host. Optionally, the host(or first and/or second bacteria) is a Erwinia host. Optionally, thehost (or first and/or second bacteria) is a Legionella (eg, pneumophila)host. Optionally, the host (or first and/or second bacteria) is aListeria (eg, monocytogenes) host. Optionally, the host (or first and/orsecond bacteria) is a Campylobacter (eg, jejuni) host. Optionally, thehost (or first and/or second bacteria) is a Yersinia (eg, pestis) host.Optionally, the host (or first and/or second bacteria) is a Borelia (eg,burgdorferi) host. Optionally, the host (or first and/or secondbacteria) is a Helicobacter (eg, pylori) host. Optionally, the host (orfirst and/or second bacteria) is a Clostridium (eg, dificile orbotulinum) host. Optionally, the host (or first and/or second bacteria)is a Erlichia (eg, chaffeensis) host. Optionally, the host (or firstand/or second bacteria) is a Salmonella (eg, typhi or enterica, eg,serotype typhimurium, eg, DT 104) host. Optionally, the host (or firstand/or second bacteria) is a Chlamydia (eg, pneumoniae) host.Optionally, the host (or first and/or second bacteria) is aParachlamydia host. Optionally, the host (or first and/or secondbacteria) is a Corynebacterium (eg, amycolatum) host. Optionally, thehost (or first and/or second bacteria) is a Klebsiella (eg, pneumoniae)host. Optionally, the host (or first and/or second bacteria) is aEnterococcus (eg, faecalis or faecim, eg, linezolid-resistant) host.Optionally, the host (or first and/or second bacteria) is aAcinetobacter (eg, baumannii, eg, multiple drug resistant) host.

In an example, the cell is a prokaryotic cell. In an example, the cellis a bacterial cell. In an example, the cell is a archaeal cell. In anexample, the cell is a microbe cell. In an example, the cell is aprotozoan cell. In an example, the cell is a fish cell. In an example,the cell is a bird cell. In an example, the cell is a reptilian cell. Inan example, the cell is an arachnid cell. In an example, the cell is ayeast cell (eg, a Saccharomyces cell). In an example, the host cell is aplant cell. In an example, the host cell is an animal cell (eg, not ahuman cell, eg, not a rodent cell). In an example, the host cell is ahuman cell (eg, not a cell in an embryo or in a human), for example ahost cell in vitro. In an example, the cell is a livestock or companionpet animal cell (eg, a cow, pig, goat, sheep, horse, dog, cat or rabbitcell). In an example, the host cell is an insect cell (an insect at anystage of its lifecycle, eg, egg, larva or pupa). In an example, the hostcell is a protozoan cell. In an example, the cell is a cephalopod cell.

Optionally the array, system, engineered nucleotide sequence or vectornucleic acid further comprises a (eg, one, tow or more) nuclearlocalisation signal (NLS), eg, for targeting to the nucleus when thehost cell is a eukaryotic cell, eg, a plant or animal. In an example, aNLS flanks each end of a Cas-encoding nucleic acid sequence of theinvention and/or an array of the invention—particularly for use intargeting in a eukaryotic host cell.

A tracrRNA sequence may be omitted from a array or vector of theinvention, for example for Cas systems of a Type that does not usetracrRNA.

In an example, the Cas guided to the target is an exonuclease.Optionally a nickase as mentioned herein is a doube nickase.

An example of a nickase is a Cas9 nickase, ie, a Cas9 that has one ofthe two nuclease domains inactivated—either the RuvC and/or HNH domain.

Optionally the host system is a Type I system (and optionally the array,HM-crRNA or gRNA is of a different CRISPR system, eg, Type II or III).Optionally the array or engineered sequence is in combination in a virusor plasmid with a nucleotide sequence encoding a Cas of the same systemas the array, HM-crRNA or gRNA, eg, where the Cas does not operate oroperate efficiently with the host system. Optionally the host system isa Type II system (and optionally the array, HM-crRNA or gRNA is of adifferent CRISPR system, eg, Type I or III). Optionally the array orengineered sequence is in combination in a virus or plasmid with anucleotide sequence encoding a Cas of the same system as the array,HM-crRNA or gRNA, eg, where the Cas does not operate or operateefficiently with the host system. Optionally the host system is a TypeIII system (and optionally the array, HM-crRNA or gRNA is of a differentCRISPR system, eg, Type I or II). Optionally the array of engineeredsequence is in combination in a virus or plasmid with a nucleotidesequence encoding a Cas of the same system as the array, eg, where theCas does not operate or operate efficiently with the host system.

Mention herein of using vector DNA can also in an alternative embodimentapply mutatis mutandis to vector RNA where the context allows. Forexample, where the vector is an RNA vector. All features of theinvention are therefore in the alternative disclosed and to be read as“RNA” instead of “DNA” when referring to vector DNA herein when thecontext allows. In an example, the or each vector also encodes a reversetranscriptase.

In an example, the or each array or engineered nucleotide sequence isprovided by a nanoparticle vector or in liposomes.

In an example, the Cas is a Cas nuclease for cutting, dead Cas (dCas)for interrupting or a dCas conjugated to a transcription activator foractivating the target.

In an example, the host CRISPR/Cas system comprises a host CRISPR arrayand a cognate host Cas for nucleotide sequence targeting in the host. Inan example, the host target sequence comprises at least 5, 6, 7, 8, 9,10, 20, 30 or 40 contiguous nucleotides. In an example, the targetsequence is cut by Cas, eg, a Cas9. In an embodiment, the sequence isnot in a spacer.

In an example, the or each array or engineered sequence comprises anexogenous promoter functional for transcription of the crRNA or gRNA inthe host.

In an example, the or each array repeats are identical to repeats in thehost array, wherein the CRISPR array does not comprise a PAM recognisedby a Cas (eg, a Cas nuclease, eg, Cas9) of the host CRISPR/Cas system.This applies mutatis mutandis to repeat sequence of the HM-crRNA andgRNA. This embodiment is advantageous since it simply enables the CRISPRarray to use the endogenous host Cas to target the host target sequence.This then is efficient as the array is tailored for use by the hostmachinery, and thus aids functioning in the host cell. Additionally, oralternatively (eg where the array is provided in combination with anexogenous (non-host endogenous) Cas-encoding sequence) this embodimentenables the CRISPR array to use the endogenously-encoded tracrRNA, sincethe CRISPR array repeats will hybridise to the endogenous tracrRNA forthe production of pre-crRNA and processing into mature crRNA thathybridises with the host target sequence. The latter complex can thenguide the endogeous Cas nuclease (eg, Cas9) or guide Cas produced fromthe sequence comprised by the CRISPR array. This embodiment thereforeprovides the flexibility of simply constructing a vector (eg, packagedvirus or phage) containing the CRISPR array but not comprising atracrRNA- and/or Cas nuclease-encoding sequence. This is morestraightforward for vector construction and also it frees up valuablespace in the vector (eg, virus or phage) which is useful bearing in mindthe capacity limitation for vectors, particularly viral vectors (eg,phage). The additional space can be useful, for example, to enableinclusion of many more spacers in the array, eg, to target the hostgenome for modification, such as to inactivate host genes or bring indesired non-host sequences for expression in the host. Additionally oralternatively, the space can be used to include a plurality of CRISPRarrays in the vector. These could, for example, be an arrangement wherea first array is of a first CRISPR/Cas type (eg, Type II or Type II-A)and the second array could be of a second type (eg, Type I or III orType II-B). Additionally or alternatively, the arrays could usedifferent Cas nucleases in the host (eg, one array is operable with thehost Cas nuclease and the second array is operable with an exogenous Casnuclease (ie, a vector-encoded nuclease)). These aspects providemachinery for targeting in the host once the vector has been introduced,which is beneficial for reducing host resistance to the vector, as thehost would then need to target a greater range of elements. For example,if the host were able to acquire a new spacer based on the first CRISPRarray sequence, the second CRISPR array could still function in the hostto target a respective target sequence in the host cell. Thus, thisembodiment is useful to reduce host adaptation to the vector.

Another benefit is that it is possible (for example, with thisarrangement) to include in the CRISPR array (or distributed over aplurality of such arrays in the vector) multiple copies of the samespacer (eg, a spacer used to target a target site in the host cell).This is beneficial since it has been proposed that adaptation of hosts,such as bacteria and archaea, may involve loss of spacers from theirarrays where the spacers target beneficial host DNA (PLoS Genet. 2013;9(9):e1003844. doi: 10.1371/journal.pgen.1003844. Epub 2013 Sep. 26,“Dealing with the evolutionary downside of CRISPR immunity: bacteria andbeneficial plasmids”, Jiang W et al). It is thought that the removal ofspacer-repeat units occurs through recombination of repeat sequences.Thus, according to the present aspect of the invention, there isprovided one, two, three, four, five, six or more CRISPR arrays orengineered sequences of the invention comprising a plurality (eg, 2, 3,4 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 ormore) copies of a spacer for hybridising to a host target sequence. Thisreduces the chances of all of these spacers being lost by recombinationin the host cell. In a further application of this aspect, the CRISPRarrays comprise a first array comprising one or more (eg, 2, 3, 4 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or more) of thespacer copies and a second array comprising one or more (eg, 2, 3, 4 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 or more) of theidentical spacer copies, wherein spacer copies in the first array areeach flanked by first repeats and the identical spacer copies in thesecond array are each flanked by second repeats, wherein the firstrepeats are different from the second repeats. This has the benefit thatat least one of the first and second repeats can be selected not to berecognised by a host Cas nuclease (or the same host Cas nuclease), toreduce the chances of host adaptation involving more than one of thearrays. In an example, the first array is in combination with a Casnuclease sequence that is not encoded by the host cell and which is acognate Cas for the first repeats. Optionally, also the second array isin combination with a Cas nuclease sequence (eg, the same or differentfrom that for the first array) that is not encoded by the host cell andwhich is a cognate Cas for the second repeats.

An embodiment provides a first array contained in a first vector and asecond array contained in a second vector which does not contain thefirst array (eg, wherein the vectors are plasmids or virions (eg, of thesame virus type) or packaged phage (eg, of the same phage type). This isuseful since the vectors can be simultaneously or sequentiallyintroduced into the same host cell. Thus, when the host gains resistanceto the first array, the second is introduced to provide a second arraywith which the resistant host (eg, bacterium or archaeon) has notpreviously co-evolved, thereby providing a second modification (eg,knock-down) wave against the host cell. This also provides flexibilitysince a third such vector, comprising a spacer or array that isdifferent from the first and second arrays and spacers, can beintroduced into the host cell simultaneously or sequentially with thesecond vector to provide a further route to host cell modification thathas not previously been present during evolution of the hosts that areresistant to a spacer in the first array. Instead of arrays, engineerednucleotide sequences of the invention can be used.

Thus, in one embodiment, the invention provides a composition formodifying a host cell, wherein the composition provides any array orengineered sequence as described herein. Thus, in one embodiment, theinvention provides a composition for modifying a host cell, wherein thecomposition provides a first array as described herein in a first vector(eg, virion or packaged phage) and a second first such array asdescribed herein in a second vector (eg, virion or packaged phagerespectively), wherein the second array comprises one or more spacersthat target one or more host target sequences and which is/are notcomprised by the first array. Instead of arrays, engineered nucleotidesequences of the invention can be used.

In an embodiment the array or engineered sequence is contained in avirophage vector and the host is alternatively a virus which can infecta cell. For example, the host is a large virus that may have infected anamoeba cell. For example, the host is a Sputnik virus, Pithovirus,mimivirus, mamavirus, Megavirus or Pandoravirus, eg, wherein the hostvirus is in water. In an example of this embodiment, the invention isfor water or sewage treatment (eg, purification, eg, waterway, river,lake, pond or sea treatment).

In an embodiment the or each vector or engineered sequence is or iscomprised by a DNM1 phage, eg, wherein the host cell(s) is a S. aureus(eg, MRSA) cell. The general features also provide the followingclauses:—

1. An antimicrobial composition (eg, an antibiotic, eg, a medicine,disinfectant or mouthwash), comprising an array, engineered sequence,virus, virion, phage, phagemid, prophage, population or collectionaccording to any aspect of the invention.

2. The composition of clause 1 for medical or dental or ophthalmic use(eg, for treating or preventing an infection in an organism or limitingspread of the infection in an organism.

In an example, the organism is a plant or animal, eg, vertebrate (eg,any mammal or human disclosed herein) or crop or food plant.

3. A composition comprising an array, engineered sequence, system,collection, virus, virion, phage, phagemid, prophage, composition,population, collection, use or method according to the invention forcosmetic use (eg, use in a cosmetic product, eg, make-up), or forhygiene use (eg, use in a hygiene product, eg, soap).

4. Use of a composition comprising an array, engineered sequence,collection, virus, virion, phage, phagemid, prophage, population orcollection according to any one of clauses 1 to 35, in medicine or fordental therapeutic or prophylacticc use.

5. Use of a composition comprising an array, engineered sequence,collection, system, virus, virion, phage, phagemid, prophage,composition, population, collection, use or method according to theinvention, in cosmetic use (eg, use in a cosmetic product, eg, make-up),or for hygiene use (eg, use in a hygiene product, eg, a soap).

6. Use of an array, engineered sequence, system, collection, virus,virion, phage, phagemid, prophage, composition, population or collectionaccording to the invention in a host modifying (HM) CRISPR/Cas9 system(eg, Type I, II or III) that is capable of modifying a target nucleotidesequence of a host cell, wherein the array, engineered sequence, system,virus, virion, phage, phagemid, prophage, population or collection isaccording to the present invention.

7. The use of clause 4, 5 or 6, wherein the array, engineered sequence,system, collection, virus, virion, phage, phagemid, prophage, populationor collection is not in a host cell.

8. The use of clause 5 or 6, wherein the array, engineered sequence,collection, system, virus, virion, phage, phagemid, prophage, populationor collection is in a host cell (eg, a microbe, bacterium or archaeoncell).

9. The use of any one of clauses 4 to 6 for modifying a microbial cell(eg, for killing or reducing growth of the cell or a culture of microbecells).

10. A method of modifying a target nucleotide sequence in a host cell(eg a microbe bacterium or archaeon), the method comprising transformingthe host cell with the array, engineered sequence, system, collection,virus, virion, phage, phagemid, population or collection according tothe present invention, whereby the target nucleotide sequence is Casmodified, wherein the host target sequence is a nucleotide sequence of ahost CRISPR/Cas system of the cell.

11. A method of reducing the development of host cell resistance totransformation by a nucleic acid vector or maintenance of a nucleic acidvector in the host cell, wherein the host cell comprises a targetnucleotide sequence, the method comprising transforming the host cellwith the array, engineered sequence, collection, system, virus, virion,phage, phagemid, population or collection according to the invention,whereby the target nucleotide sequence is Cas modified (eg, cut, mutatedor knocked-down).

12. The method of clause 11, wherein the vector is a virus that iscapable of infecting the host cell and the transforming step comprisesinfecting the host cell with the vector.

13. The method of clause 11 or 12, wherein the host cell is a bacterialor archaeal cell and the vector is a phage or phagemid.

14. The method of any one of clauses 11 to 13, wherein the host targetsequence is essential to host CRISPR/Cas-mediated acquisition of vectorsequence spacers.

15. The array, engineered sequence, system, vector, cell, collection,composition, use or method of any preceding clause, wherein at leastcomponent (ii) is contained in a virus (eg, a phage) that is capable ofexpressing an endolysin for host cell lysis, optionally wherein theendolysin is a phage phi11, phage Twort, phage P68, phage phiWMY orphage K endolysin (eg, MV-L endolysin or P-27/HP endolysin).

16. The array, engineered sequence, system, vector, collection, cell,composition, use or method of clause 15 in combination with an endolysinfor host cell lysis, eg, in combination with MV-L endolysin or P-27/HPendolysin or a functional homologue thereof.

17. The array, engineered sequence, system, vector, collection, cell,composition, use or method of any preceding clause in combination withan antimicrobial, eg, antibiotic agent, eg, a beta-lactam antibiotic.

18. The array, engineered sequence, system, vector, collection, cell,composition, use or method of any preceding clause, wherein the hostcell is a Staphylococcus, Streptococcus, Pseudomonas, Salmonella,Listeria, E coli, Desulfovibrio or Clostridium host cell.

19. The array, engineered sequence, system, vector, collection, cell,composition, use or method of any preceding clause, wherein the hostcell is a Staphylococcus (eg, S aureus) host cell and at least component(ii) is contained in a Class I, II or III Staphylococcus phage (eg, apackaged phage), optionally a Caudovirales or Myoviridae phage.

20. The array, engineered sequence, system, vector, cell, collection,composition, use or method of any preceding clause, wherein the hostcell is a beta-lactam antibiotic-resistant Streptococcus aureus,methicillin-resistant Streptococcus aureus (MRSA), vancomycin-resistantStreptococcus aureus or teicoplanin-resistant Streptococcus aureus andoptionally the target sequence is a sequence of a host beta-lactamantibiotic-resistance gene, methicillin-resistance gene,vancomycin-resistance gene or teicoplanin-resistance gene respectively.

Suitable methods for producing and testing phage vectors of theinvention are, for example, general methods disclosed in WO2014/124226.

Mobile Genetic Elements, Transposons & Carriers (for any Configurationof the Invention)

Plasmids are very common in Bacteroides species and are found in 20 to50% of strains. Many plasmids possess oriT and a transactingmobilisation gene, which allow them to be transferred by conjugation.Thus, in an example, the vector is a plasmid comprising oriT and/or amobilisation gene, eg, wherein the first or second bacteria areBacteroides. In an example, the engineered sequence is comprised by sucha vector.

In an example, the host cells, or the first or second bacteria naturallycomprise transposons. Transposons, both mobilisable and conjugative, donot replicate independently; rather, they excise from and integrate intochromosomal DNA and are copied along with the chromosomal DNA.Conjugative transposons have a mechanism of excision and integrationthat resemble some features of both plasmids and bacteriophage.Conjugative transposons are practically ubiquitous among theBacteroides: over 80% of Bacteroides strains contain at least oneconjugative transposon. The conjugative transposons of Bacteroidesbelong to at least two families; CTnDot is the best described. Often,the name of the strain in which they are found is added to thedesignation (e.g., CTnDot, found in the DOT strain of Bthetaiotaomicron). In addition to being able to insert into thechromosome, Bacteroides conjugative transposons can insert intocoresident plasmids and mobilise them in cis (i.e., they can act onentities that are physically adjacent) by integrating themselves intothe plasmid and facilitating transfer of the plasmid-conjugativetransposon hybrid into another cell. They can also mobilise coresidentplasmids “in trans” by supplying factors needed to facilitate transferof the plasmid, while remaining physically separate from the plasmid.

Conjugative transposons do not exclude each other as do plasmids, so astrain can accumulate more than one conjugative transposon. Furthermore,there is some evidence that the presence of more than one copy of theconjugative transposon in the strain results in a stimulation oftransposition (transactivation). Theoretically, this suggests that asmore conjugative transposons accumulate in the environment, the transferof the transposon genes to other bacteria will also increase, and therewill be a significant upward spiraling of distribution of the genes.Many of the Bacteroides transposons carry the tetQ gene and thus confertetracycline resistance. Further, self-transfer and other activities aresignificantly stimulated by low levels of tetracycline, regulated by thetetQ-rteA-rteB operon. Tetracycline increases transcription of rteA and-B, which code for the sensor and activator components of atwo-component regulatory system. In turn, RteB activates expression ofrteC, which is necessary for self-transfer.

In an example, the vector (eg, the vector comprising the engineeredsequence) comprises a transposon, wherein the transposon comprises theengineered sequence, HM- or PM-array of the invention, wherein thetransposon is operable in the host cell(s) or in the first or secondbacteria host cell species. In an embodiment, the transposon is aBactroides transposon (eg, a CTnDot transposon, eg, a B thetaiotaomicronor B fragalis CTnDot tranposon) and the host cells, or the first orsecond bacteria are or comprise Bacteroides (eg, of the same species assaid CTnDot transposon). In an example, the transposon is a conjugativetransposon. In an example, the transposon is a mobilisable transposon.In an example, the transposon is transferable between Bacteroides cells.In an example, the transposon comprises an intDOT sequence. In anexample, the transposon comprises an oriT. In an example, the transposonencodes one or more mating pore proteins for conjugative transfer of thetransposon between host cells.

In an example, the invention provides a transposon that comprises aBacteroides tetQ gene. In an example, the transposon further comprises aBacteroides tetQ-rteA-rteB operon. In an example, the first or secondbacteria are Bacteroides. In an example, the transposon is a BacteroidesCTnDot transposon that encodes one or more mating pore proteins forconjugative transfer of the transposon between host cells and comprisesone or more arrays or engineered sequences of the invention, an oriT, anintDOT sequence and a tetQ-rteA-rteB operon, and is optionally foradministration or is administered to said human or non-human animal asmentioned herein in combination with tetracycline. Transfer of mostBacteroides CTns is stimulated by tetracycline. The transposon isoperable with an integrase in a host cell or is operable with anexogenous integrase carried on the same or a different vector to thetransposon. In an embodiment, the vector is a phage (eg, a packagedphage) comprising the transposon and a nucleotide sequence encoding acognate integrase. The phage is capable of infecting a host bacterialcell for replication and excision of the transposon, eg, for conjugativetransfer to neighbouring host cells in a mixed bacterial population (eg,a gut microbiota population).

In an embodiment, the transposon is comprised by a vector that carriesone or more gene sequences necessary for transposon transfer betweenhost cells, wherein said gene sequences are outside of the transposon onthe vector nucleic acid. For example, the vector is a packaged phagethat is capable of infecting a host cell (eg, a Bacteroides host cell),wherein the phage nucleic acid comprises a said transposon comprising aarray of the invention and upstream or downstream of the transposon oneor more genes operable for conjugative transfer of the transposon (eg,one or more genes encoding relaxes, coupling proteins and/or matingbridge proteins for transposon conjugative transfer; and/or one or bothof mob and tra operons), wherein one, more or all these genes is notcomprised by the transposon. In an example, these genes are genes forexcision of the transposon from chromosomal DNA inside a first hostcell. Thus, the transposon is able to mobilise inside that cell andcarries with it genes necessary for the subsequent conjugative transferinto a second host cell. By providing some of the transposon genes inthis way on the vector outside the transposon, this frees up room in thetransposon for inclusion of engineered sequence or array DNA of theinvention so that this can be accommodated and carried by mobilisedtransposons. The invention provides such a vector comprising one or moresuch transposons for use in the method, use or system of the inventionor generally for introduction into bacterial cells (in this case insteadof targeting a phage sequence, the array included in the transposon cantarget a bacterial target sequence to modify the sequence, eg, cut itusing Cas in a cell harbouring the transposon).

Molecular mechanisms of CTnDot excision and integration more closelyresemble that of bacteriophage rather than transposition. The CTnDOTintegrase and excision proteins themselves are quite similar to thosefrom bacteriophage. Thus, in one embodiment the function of one or moreintegrase and/or excision proteins of the transposon of the inventionare provided by the phage integrase and/or excision proteinsrespectively, and the transposon does not comprise corresponding gene(s)encoding such integrase or excision proteins whose functions areprovided by phage proteins.

In an example, the transposon comprises rteC and the operonxis2c-xis2d-orf3-exc. Optionally, additionally the vector comprises moband tra operons outside of the transposon (eg, upstream or downstream ofthe transposon in the vector nucleic acid). Thus, this frees up space inthe transposon for providing CRISPR array sequence or engineeredsequence of the invention.

Many conjugative transposons are able to mobilise other elements. Forexample, many coresident plasmids are mobilized by a conjugativetransposon in trans. This occurs when a plasmid containing an oriTutilizes the CTn-provided mating pore proteins for transfer to arecipient cell. The Bacteroides CTns have also been shown to mobilizeelements when in cis, a feature that is not typical for CTns. Forexample, if CTnDOT excises from the chromosome and integrates on aplasmid, it can provide the mating pore, an oriT, and the mobilization(relaxase/coupling) proteins, allowing it to transfer the entire plasmidby acting “in cis.” This ability to use both trans and cis mechanisms ofmobilization is unusual and suggests that the Bacteroides CTns have agreater capacity to mobilize other elements.

In an example, the vector of the invention is a plasmid comprising oneor more engineered sequences or arrays of the invention and an oriT thatis cognate to a host cell species CTnDot transposon that encodes matingpore proteins, whereby the plasmid is mobilisable in a host cellcomprising a said CTnDot transposon. Thus, the plasmid is capable ofhorizontal transfer between host cells, thereby spreading arrays of theinvention in a population of such host cells (eg, Bacteroides cells). Inan example, the invention provides a composition comprising a populationof carrier bacteria, wherein the carrier bacteria are compatible withsuch a plasmid vector of the invention, whereby the vector is capable ofhorizontal transfer to recipient host bacteria cells (eg, Bacteroides orFirmicutes, eg, Streptococcus cells) comprising cognate CTnDottransposons when the carrier and recipient bacteria are mixed. In anexample, the carrier bacteria are comprised by a beverage (eg, probioticdrink, such as one described herein) or foodstuff for human or non-humananimal consumption, whereby the carrier bacteria can mix with recipientbacteria harboured by the human or animal (eg, in the oral cavity or inthe gut). Other transposons within the CTnDOT-like family include CTnERLand CTn341, although these elements differ from CTnDOT, and thus insteadof a CTnDot transposon, the transposon of the general aspect of theinvention can be a CTnERL or CTn341 transposon carrying one or moredesired CRISPR arrays or engineered sequences for targeting one or morebacterial or phage nucleotide target sites when the transposon iscomprised by a bacterial or archaeal host cell.

In order for transfer of the conjugative transposon to occur, there arethree main steps that take place. The first step is excision from thechromosome to form a covalently closed circular intermediate. Second, asingle-stranded copy is then transferred through the mating pore to arecipient cell, after which the copy becomes double stranded. Third, theintact double-stranded CTn integrates into the chromosome of therecipient. Conjugative transposition is replicative, as a copy of theCTn is retained in the donor cell. Because the element resides withinthe chromosome, it is also transferred vertically to progeny cells. Thisis important because when desired CRISPR arrays or engineered sequences(and optionally Cas sequence) are present on CTns, they are not onlytransferred readily within the population, but they are also very stablymaintained from generation to generation. This is as seen, for example,with retained antibiotic resistance determinants. Further, it isbelieved that Bacteroides may serve as a reservoir of antibioticresistance determinants which disseminates these genes to otherorganisms outside the Bacteroides genus, possibly even transferringthese elements to organisms that are transiently passing through thegut. Similarly, a reservoir of arrays or engineered sequences of theinvention can be created using vectors of the invention that areadministered to a human or non-human animal, eg, for treating orpreventing obesity, diabetes or IBD or any other disease or conditiondisclosed herein.

In an example, one can exploit the reservoir of desired CRISPR arrays orengineered sequences by using one or more arrays or sequences comprisedby a transposon (eg, a CTnDot) that is capable of being harboured byBacteroides cells (eg, in the gut or oral cavity of a human or non-humananimal), wherein the array(s)/sequence(s) do not target a sequence ofthe host Bacteroides cell, but do target a nucleotide sequence comprisedby a gut microbiota cell (eg, bacterial cell) of a different species(eg, a Firmicutes cell or pathogenic bacterial cell, eg, Streptococcus,C dificile, H pylori, Salmonella, Listeria, Yersinia, Shigella orCampylobacter cell). Thus, in this way transfer of the arrays orsequences of the invention to neighbouring recipient pathogenic orundesired bacteria can take place, and once inside the recipient cellthe array(s) of the invention are operable to guide Cas to therespective target site in the host cell to modify (eg, cut) the site. Inthis case, the array/sequence can comprise repeat sequences that arefound in the recipient cell of interest so that the array/sequence canoperate with an endogenous CRISPR/Cas system inside the recipient cell.This avoids the need to include Cas and/or tracrRNA-encoding sequencesin the vector, engineered sequence or transposon of the invention,thereby freeing up space and simplifying construction. Increased spaceis useful for enabling inclusion of more spacers to target more targetsites in the recipient cell. In an alternative, the transposon array(s)or sequence(s) comprises a Type II Cas9-encoding sequence and cognaterepeat sequences. For example, the Cas9 (any Cas9 mentioned herein) is aS pyogenes, S thermophilus or S aureus Cas9 and may optionally be anickase or dCas9 (“dead Cas9”). As Bacteroides are obligate anaerobes(or have a strong preference for anaerobic environments) and typicallyare pathogenic outside the gut environment, it may not be desirable touse Bacteroides cells as carriers for the vectors or transposons of theinvention, eg, when administering to the gut or oral cavity of a humanor animal. To address this, the invention provides a carrier populationof bacteria harbouring vectors, engineered sequence(s) or transposons ofthe invention, wherein the carrier bacteria are compatible with such avector, sequence or transposon, whereby the vector, sequence ortransposon is capable of horizontal transfer to recipient host bacteriacells (eg, Bacteroides) in gut microbiota when the carrier and recipientbacteria are mixed. In an example, the carrier bacteria are comprised bya beverage (eg, probiotic drink, such as one described herein) orfoodstuff for human or non-human animal consumption, whereby the carrierbacteria can mix with recipient bacteria harboured by the human oranimal (eg, in the oral cavity or in the gut). In an embodiment, thevectors, sequences or transposons comprise CRISPR arrays of theinvention, wherein the arrays target nucleotide sequences comprised bythe recipient cells to modify the target sequences, eg, by cutting thesequences to inactivate genes comprising the target sequences. In analternative, the vectors, sequences or transposons are capable ofhorizontal transfer (eg, conjugative transposon transfer) to a secondrecipient population of bacteria, which are of a different species tothe first recipient bacteria, wherein the nucleotide sequence targetsites are comprised by the second recipient bacteria but not comprisedby the first recipient bacteria, whereby the target sites are modifiedby Cas in the second recipient bacteria (host cells).

In an example, the first recipient bacteria are Bacteroides bacteria andthe second recipient bacteria are Firmicutes or pathogenic bacteria, eg,gut bacteria. In an example, the carrier bacteria comprise vectors ofthe invention (eg, phage or plasmids) comprising one or more conjugativetransposons (eg, CTnDot transposons) that are capable of being harbouredby the carrier bacteria, first bacteria and second bacteria, eg, whereinthe transposons comprise oriT and the carrier bacteria, first bacteriaand second bacteria are compatible with oriT.

In an alternative, the carrier bacteria are capable of transferring thevector, engineered sequence or transposon of the invention directly toFirmicutes or pathogenic bacteria, eg, in an animal or non-human animal,eg, in the gut, oral cavity or systemically (eg, in the blood). In anexample, the pathogenic bacteria are C dificile, H pylori, pathogenic Ecoli, Salmonella, Listeria, Yersinia, Shigella, S aureus, Streptococcusor Campylobacter bacteria.

In an example, the carrier bacteria are bacteria of one or more speciesselected from the group consisting of a Lactobacillus species (eg,acidophilus (eg, La-5, La-14 or NCFM), brevis, bulgaricus, plantarum,rhammosus, fermentum, caucasicus, helveticus, lactis, reuteri or caseieg, casei Shirota), a Bifidobacterium species (eg, bifidum, breve,longum or infantis), Streptococcus thermophilus and Enterococcusfaecium. For example, the bacteria are L acidophilus bacteria.

Mobilisable transposons, like mobilisable plasmids, cannot self-transferbut can transfer between cells in the presence of the TcR helperelement. The most commonly discussed Bacteroides transposons of thisclass include Tn4399, Tn4555, and the nonreplicating Bacteroides units.The mobilisable transposon Tn4555, for example, was first detectedduring studies of transmissible cefoxitin resistance in a clinicalisolate of Bacteroides vulgatus. In an embodiment, therefore, thetransposon of the invention is a mobilisable transposon (eg, aBacteroides mobilisable transposon), eg, a Tn4399 or Tn4555 comprisingone or more arrays or sequences of the invention. The transposon is incombination with a TcR helper element.

In an example, the transposon of the invention is Enterococcus Tn916 orGram-positive Tn1546 transposon. A transposon (eg, as a CTnDot, Tn4399or Tn4555 transposon) can be characterised for example according to itsterminal repeats and/or transposase- or resolvase-encoding sequence(s).In an alternative example, the vector or transposon comprises an originof replication selected from pMB 1, pBR322, ColE1, R6K (in combinationwith a pir gene), p15A, pSC101, F1 and pUC. In an example, thetransposon is in combination with a factor (eg, an antibiotic, eg,tetracycline) that is required for transposon mobilisation or transfer.In an example, the transposon comprises an antibiotic resistance gene(eg, tetracycline resistance gene) and the transposon is in combinationwith said antibiotic (eg, administered simultaneously or sequentially tothe human with said antibiotic). In an example, the transposon is apiggyBac, Mariner or Sleeping Beauty transposon in combination with acognate transposase. In an example, the transposon is a Class Itransposon. In an example, the transposon is a Class II transposon. Inan example, the transposon is a Tn family transposon.

Targeting Antibiotic Resistance in Bacterial Hosts

Antibiotic resistance is a worldwide problem. New forms of antibioticresistance can cross international boundaries and spread betweencontinents with ease. Many forms of resistance spread with remarkablespeed. World health leaders have described antibiotic resistantmicroorganisms as “nightmare bacteria” that “pose a catastrophic threat”to people in every country in the world. Each year in the United States,at least 2 million people acquire serious infections with bacteria thatare resistant to one or more of the antibiotics designed to treat thoseinfections. At least 23,000 people die each year as a direct result ofthese antibiotic-resistant infections. Many more die from otherconditions that were complicated by an antibiotic resistant infection.In addition, almost 250,000 people each year require hospital care forClostridium difficile (C. difficile) infections. In most of theseinfections, the use of antibiotics was a major contributing factorleading to the illness. At least 14,000 people die each year in theUnited States from C. difficile infections. Many of these infectionscould have been prevented. Antibiotic-resistant infections addconsiderable and avoidable costs to the already overburdened U.S. andother healthcare systems. In most cases, antibiotic-resistant infectionsrequire prolonged and/or costlier treatments, extend hospital stays,necessitate additional doctor visits and healthcare use, and result ingreater disability and death compared with infections that are easilytreatable with antibiotics. The total economic cost of antibioticresistance to the U.S. economy has been difficult to calculate.Estimates vary but have ranged as high as $20 billion in excess directhealthcare costs, with additional costs to society for lost productivityas high as $35 billion a year (2008 dollars). The use of antibiotics isthe single most important factor leading to antibiotic resistance aroundthe world. Antibiotics are among the most commonly prescribed drugs usedin human medicine. However, up to 50% of all the antibiotics prescribedfor people are not needed or are not optimally effective as prescribed.Antibiotics are also commonly used in food animals to prevent, control,and treat disease, and to promote the growth of food-producing animals.The use of antibiotics for promoting growth is not necessary, and thepractice should be phased out. Recent guidance from the U.S. Food andDrug Administration (FDA) describes a pathway toward this goal. It isdifficult to directly compare the amount of drugs used in food animalswith the amount used in humans, but there is evidence that moreantibiotics are used in food production.

The other major factor in the growth of antibiotic resistance is spreadof the resistant strains of bacteria from person to person, or from thenon-human sources in the environment, including food. There are fourcore actions that will help fight these deadly infections: 1. preventinginfections and preventing the spread of resistance; 2. trackingresistant bacteria; 3. improving the use of today's antibiotics; and 4.promoting the development of new antibiotics and developing newdiagnostic tests for resistant bacteria. Bacteria will inevitably findways of resisting the antibiotics we develop, which is why aggressiveaction is needed now to keep new resistance from developing and toprevent the resistance that already exists from spreading.

The invention provides improved means for targeting antibiotic-resistanthosts and for reducing the likelihood of hosts developing furtherresistance to the compositions of the invention.

Further examples of host cells and targeting of antibiotic resistance insuch cells using the present invention are as follows:—

1. Optionally the host cell(s) are Staphylococcus aureus cells, eg,resistant to an antibiotic selected from methicillin, vancomycin,linezolid, daptomycin, quinupristin, dalfopristin and teicoplanin andthe host target site (or one or more of the target sites) is comprisedby a gene conferring host resistance to said antibiotic.

2. Optionally the host cell(s) are Pseudomonas aeuroginosa cells, eg,resistant to an antibiotic selected from cephalosporins (eg,ceftazidime), carbapenems (eg, imipenem or meropenem), fluoroquinolones,aminoglycosides (eg, gentamicin or tobramycin) and colistin and the hosttarget site (or one or more of the target sites) is comprised by a geneconferring host resistance to said antibiotic.

3. Optionally the host cell(s) are Klebsiella (eg, pneumoniae) cells,eg, resistant to carbapenem and the host target site (or one or more ofthe target sites) is comprised by a gene conferring host resistance tosaid antibiotic.

4. Optionally the host cell(s) are Streptococcus (eg, thermophilus,pneumoniae or pyogenes) cells, eg, resistant to an antibiotic selectedfrom erythromycin, clindamycin, beta-lactam, macrolide, amoxicillin,azithromycin and penicillin and the host target site (or one or more ofthe target sites) is comprised by a gene conferring host resistance tosaid antibiotic.

5. Optionally the host cell(s) are Salmonella (eg, serotype Typhi)cells, eg, resistant to an antibiotic selected from ceftriaxone,azithromycin and ciprofloxacin and the host target site (or one or moreof the target sites) is comprised by a gene conferring host resistanceto said antibiotic.

6. Optionally the host cell(s) are Shigella cells, eg, resistant to anantibiotic selected from ciprofloxacin and azithromycin and the hosttarget site (or one or more of the target sites) is comprised by a geneconferring host resistance to said antibiotic.

7. Optionally the host cell(s) are mycobacterium tuberculosis cells, eg,resistant to an antibiotic selected from Resistance to isoniazid (INH),rifampicin (RMP), fluoroquinolone, amikacin, kanamycin and capreomycinand azithromycin and the host target site (or one or more of the targetsites) is comprised by a gene conferring host resistance to saidantibiotic.

8. Optionally the host cell(s) are Enterococcus cells, eg, resistant tovancomycin and the host target site (or one or more of the target sites)is comprised by a gene conferring host resistance to said antibiotic.

9. Optionally the host cell(s) are Enterobacteriaceae cells, eg,resistant to an antibiotic selected from a cephalosporin and carbapenemand the host target site (or one or more of the target sites) iscomprised by a gene conferring host resistance to said antibiotic.

10. Optionally the host cell(s) are E. coli cells, eg, resistant to anantibiotic selected from trimethoprim, itrofurantoin, cefalexin andamoxicillin and the host target site (or one or more of the targetsites) is comprised by a gene conferring host resistance to saidantibiotic.

11. Optionally the host cell(s) are Clostridium (eg, dificile) cells,eg, resistant to an antibiotic selected from fluoroquinolone antibioticand carbapenem and the host target site (or one or more of the targetsites) is comprised by a gene conferring host resistance to saidantibiotic.

12. Optionally the host cell(s) are Neisseria gonnorrhoea cells, eg,resistant to an antibiotic selected from cefixime (eg, an oralcephalosporin), ceftriaxone (an injectable cephalosporin), azithromycinand tetracycline and the host target site (or one or more of the targetsites) is comprised by a gene conferring host resistance to saidantibiotic.

13. Optionally the host cell(s) are Acinetoebacter baumannii cells, eg,resistant to an antibiotic selected from beta-lactam, meropenem and acarbapenem and the host target site (or one or more of the target sites)is comprised by a gene conferring host resistance to said antibiotic.

14. Optionally the host cell(s) are Campylobacter cells, eg, resistantto an antibiotic selected from ciprofloxacin and azithromycin and thehost target site (or one or more of the target sites) is comprised by agene conferring host resistance to said antibiotic.

15. Optionally, the host cell(s) produce Beta (13)-lactamase.

16. Optionally, the host cell(s) are bacterial host cells that areresistant to an antibiotic recited in any one of examples 1 to 14.

In an embodiment, the host cell is a USA300 S aureus strain cell.

In an example, the or each host target sequence is comprised by aplasmid of the host cell, eg, a S aureus plasmid (eg, of a USA300strain), eg, a target comprised by the pUSA01, pUSA02 or pUSA03 plasmidof a S aureus cell. In an example, the first and/or second target iscomprised by a host mecA, mecA2 or sek gene sequence (eg, of a S aureusstrain cell). In an example, the first and/or second target is comprisedby a host pathogenicity island nucleotide (eg, DNA) sequence. Inexample, a spacer of the invention comprises or consists of a spacerdisclosed in Table 1 on page 26 of WO2014/124226, which spacer sequencesare incorporated herein by reference. In an example, the engineeredsequence, HM-crRNA or gRNA comprises such a spacer.

The composition, use, method system, vector, collection, array,engineered sequence, virus, phage, phagemid, prophage or virion of theinvention which is effective to reduce or kill or inhibit growth of anantibiotic-resistant bacterial host in a mouse skin colonisation assay(eg, as disclosed in WO2014/124226, Kugelberg E, et al. Establishment ofa superficial skin infection model in mice by using Staphylococcusaureus and Streptococcus pyogenes. Antimicrob Agents Chemother. 2005;49:3435-3441 or Pastagia M, et al. A novel chimeric lysin showssuperiority to mupirocin for skin decolonization ofmethicillin-resistant and -sensitive Staphylococcus aureus strains.Antimicrob Agents Chemother. 2011; 55:738-744) wherein the first and/orsecond target is comprised by a host gene that confers resistance tosaid antibiotic, eg, wherein the host is a S aureus (eg, USA300 strain)host.

Reference S pyogenes sequence is available under Genbank accessionnumber NC_002737. with the cas9 gene at position 854757-858863. The Spyogenes Cas9 amino acid sequence is available under number NP_269215.These sequences are incorporated herein by reference for use in thepresent invention. Further sequences as disclosed in 20150079680,whether explicitly or incorporated by reference therein, are alsoincorporated herein by reference for use in the present invention.Reference is also made to the disclosure of sequences and methods inWO2013/176772, which is incorporated herein by reference. ExampletracrRNA sequences are those disclosed on page 15 of WO2014/124226,which are incorporated herein by reference for use in the presentinvention.

In an example, the or each repeat comprises or consists of from 20 to 50(eg, from 24 to 47, eg, 30, 29, 28, 27, 26, 25 or 24) contiguousnucleotides in length.

In an example, the or each spacer comprises or consists of from 18 to 50(eg, from 24 to 47, or 20 to 40, eg, 30, 29, 28, 27, 26, 25, 24, 23, 22,21, 20, 19, 18), eg, 19 or 20 contiguous nucleotides in length.

In an example, the first repeat (most 5′ in the HM-array of theinvention) is immediately 5′ of a spacer sequence that is complementaryto a sequence comprising the first host target. This is useful, in viewof the observation that newly acquired spacers (eg of invading phagesequence) are commonly incorporated at this position in bacteria, andthus positioning of the first spacer of the invention in this way isuseful to promote its use.

In an example, the virus (eg, phage) nucleic acid comprises an origin ofreplication (ori) and a packaging site. In an example, the nucleic acidof the virus also comprises one, more or all genes encoding essentialcapsid proteins, eg, rinA, terS and terL genes. In an example, one, moreor all of these is instead comprised by a companion helper virus (eg,helper phage) that is for con-infection with the virus of theinvention—this frees up space in the latter for including more HM-arraynucleic acid and/or more Cas-encoding nucleic acid operable in the host.In an example, the virus nucleic acid comprises a fragment of awild-type phage genome, wherein the fragment consists of consecutivenucleotides of the genome comprising at least the rinA, terS and terLgenes or equivalent genes encoding phage proteins.

In an example, the host cell is of a strain or species found in humanmicrobiota.

In an example, the or each target site is comprised by a gene thatmediates host pathogenic adhesion, colonisation, invasion, immuneresponse inhibition, virulence, essential protein or function expressionor toxin generation. In an example, the gene is a gene encoding acytotoxin, alpha-haemolysin, beta-haemolysin, gamma-haemolysin,leukocidin, Panton-Valentine lekocidin (PVL), exotoxin, TSST-1,enterotoxin, SEA, SEB, SECn, SED, SEE, SEG, SEH, SEI, exfolative toxin,ETA or ETB, optionally wherein the host is S aureus, eg, MRSA.

In an example, the or each CRISPR array is an array according to any ofthe configurations, embodiments, examples, concepts, aspects, paragraphsor clauses disclosed herein. In an example, the or each engineerednucleotide sequence is an engineered nucleotide sequence according toany of the configurations, embodiments, examples, concepts, aspects,paragraphs or clauses disclosed herein.

In an example, the or each vector is a vector according to any of theconfigurations, embodiments, examples, concepts, aspects, paragraphs orclauses disclosed herein.

In an example according to any of the configurations, embodiments,examples, Aspects, paragraphs or clauses disclosed herein, the vector orMGE is or comprises a casposon. MGEs are described further below. In anexample, the casposon is a family 1, 2 or 3 casposon. In an example, anMGE of the invention comprises casposon terminal inverted repeats andoptionally a casposon Cas1-encoding sequence. In an example, an MGE ofthe invention is or comprises a casposon minus Cas1 and operable formobilisation with Cas1 of a host cell. See BMC Biol. 2014 May 19; 12:36.doi: 10.1186/1741-7007-12-36, “Casposons: a new superfamily ofself-synthesizing DNA transposons at the origin of prokaryoticCRISPR-Cas immunity”, Krupovic M et al for details of casposons.

Further Example Applications of the Present Invention

In an example, the composition (eg, HM-composition or engineeredsequence in combination with antibiotic) is as any of the following: Inan example, the composition is a medical, ophthalmic, dental orpharmaceutical composition (eg, comprised by a an anti-host vaccine). Inan example, the composition is a an antimicrobial composition, eg, anantibiotic or antiviral, eg, a medicine, disinfectant or mouthwash. Inan example, the composition is a cosmetic composition (eg, face or bodymake-up composition). In an example, the composition is a herbicide. Inan example, the composition is a pesticide (eg, when the host is aBacillus (eg, thuringiensis) host). In an example, the composition is abeverage (eg, beer, wine or alcoholic beverage) additive. In an example,the composition is a food additive (eg, where the host is an E coli,Salmonella, Listeria or Clostridium (eg, botulinum) host). In anexample, the composition is a water additive. In an example, thecomposition is a additive for aquatic animal environments (eg, in a fishtank). In an example, the composition is an oil or petrochemicalindustry composition or comprised in such a composition (eg, when thehost is a sulphate-reducing bacterium, eg, a Desulfovibrio host). In anexample, the composition is a oil or petrochemical additive. In anexample, the composition is a chemical additive. In an example, thecomposition is a disinfectant (eg, for sterilizing equipment for humanor animal use, eg, for surgical or medical use, or for baby feeding). Inan example, the composition is a personal hygiene composition for humanor animal use. In an example, the composition is a composition forenvironmental use, eg, for soil treatment or environmentaldecontamination (eg, from sewage, or from oil, a petrochemical or achemical, eg, when the host is a sulphate-reducing bacterium, eg, aDesulfovibrio host). In an example, the composition is a plant growthstimulator. In an example, the composition is a composition for use inoil, petrochemical, metal or mineral extraction. In an example, thecomposition is a fabric treatment or additive. In an example, thecomposition is an animal hide, leather or suede treatment or additive.In an example, the composition is a dye additive. In an example, thecomposition is a beverage (eg, beer or wine) brewing or fermentationadditive (eg, when the host is a Lactobacillus host). In an example, thecomposition is a paper additive. In an example, the composition is anink additive. In an example, the composition is a glue additive. In anexample, the composition is an anti-human or animal or plant parasiticcomposition. In an example, the composition is an air additive (eg, forair in or produced by air conditioning equipment, eg, where the host isa Legionella host). In an example, the composition is an anti-freezeadditive (eg, where the host is a Legionella host). In an example, thecomposition is an eyewash or ophthalmic composition (eg, a contact lensfluid). In an example, the composition is comprised by a dairy food (eg,the composition is in or is a milk or milk product; eg, wherein the hostis a Lactobacillus, Streptococcus, Lactococcus or Listeria host). In anexample, the composition is or is comprised by a domestic or industrialcleaning product (eg, where the host is an E coli, Salmonella, Listeriaor Clostridium (eg, botulinum) host). In an example, the composition iscomprised by a fuel. In an example, the composition is comprised by asolvent (eg, other than water). In an example, the composition is abaking additive (eg, a food baking additive). In an example, thecomposition is a laboratory reagent (eg, for use in biotechnology orrecombinant DNA or RNA technology). In an example, the composition iscomprised by a fibre retting agent. In an example, the composition isfor use in a vitamin synthesis process. In an example, the compositionis an anti-crop or plant spoiling composition (eg, when the host is asaprotrophic bacterium). In an example, the composition is ananti-corrosion compound, eg, for preventing or reducing metal corrosion(eg, when the host is a sulphate-reducing bacterium, eg, a Desulfovibriohost, eg for use in reducing or preventing corrosion of oil extraction,treatment or containment equipment; metal extraction, treatment orcontainment equipment; or mineral extraction, treatment or containmentequipment). In an example, the composition is an agricultural or farmingcomposition or comprised in such a composition. In an example, thecomposition is a silage additive. The invention provides a HM-CRISPRarray, HM-CRISPR/Cas system, HM-crRNA, HM-spacer, HM-DNA, HM-Cas,HM-composition or gRNA as described herein for use in any of thecompositions described in this paragraph or for use in any applicationdescribed in this paragraph, eg, wherein the host cell is a microbialcell or a bacterial or archaeal cell. The invention provides a methodfor any application described in this paragraph, wherein the methodcomprises combining a HM-CRISPR array, HM-CRISPR/Cas system, HM-crRNA,HM-spacer, HM-DNA, HM-Cas, gRNA or HM-composition of the invention witha host cell (eg, microbial, bacterial or archaeal cell). In anembodiment, the host cell is not present in or on a human (or humanembryo) or animal.

Any aspect of the present invention is for an industrial or domesticuse, or is used in a method for such use. For example, it is for or usedin agriculture, oil or petroleum industry, food or drink industry,clothing industry, packaging industry, electronics industry, computerindustry, environmental industry, chemical industry, aerospace industry,automotive industry, biotechnology industry, medical industry,healthcare industry, dentistry industry, energy industry, consumerproducts industry, pharmaceutical industry, mining industry, cleaningindustry, forestry industry, fishing industry, leisure industry,recycling industry, cosmetics industry, plastics industry, pulp or paperindustry, textile industry, clothing industry, leather or suede oranimal hide industry, tobacco industry or steel industry.

Herein, where there is mention of a Desulfovibrio host, the host can beinstead a Desulfobulbus, Desulfobacter, Desulfobacterium, Desulfococcus,Desulfomonile, Desulfonema, Desulfobotulus or Desulfoarculus host or anyother sulphur-reducing bacterium disclosed herein. In an embodiment foroil, water, sewage or environmental application, the host is aDesulfovibrio capillatus host. Extensive microbiological analysis and16S rRNA sequencing have indicated that the genus Desulfovibrio is butone of about eight different groups of sulfate-reducing eubacteria thatcan be isolated from the environment. Seven of these groups aregram-negative, while one represents the gram-positive bacteria(Desulfotomaculum). The genus Desulfovibrio has a rather small genome.Initial estimates were 1.7 Mbp and 1.6 Mbp for the genomes of D.vulgaris and D. gigas (which may be hosts according to the invention),respectively. This aids identification of desired target sequences (eg,a sequence in an essential or resistance gene) for use in the invention.Characterization of an indigenous plasmid of D. desulfuricans (which maybe a host according to the invention) G200 has allowed the constructionof a shuttle vector (Wall 1993, which vector may be used as a vector forthe present invention), and the isolation and characterization of twobacteriophages from D. vulgaris Hildenborough (which may be a hostaccording to the invention) (Seyedirashti, 1992) may provide other waysto efficiently genetically manipulate Desulfovibrio spp. In an example,the vector is a mu or mu-like bacteriophage.

An example host is Desulfovibrio vulgaris subsp. vulgaris Postgate andCampbell (ATCC® 29579™) strain designation: NCIB 8303 [DSM 644,Hildenborough].

Treatment of the bacteria with mitomycin C or UV has previously beenused to induce phage from the bacteria (Driggers & Schmidt), and this isa suitable method for obtaining suitable host-matched phage forgenerating a vector for use in any example or aspect of the presentinvention.

An application of the invention is in the dairy industry (eg, cheese orbutter or milk products manufacture) or fermenting (eg, wine or vinegaror soy) or beer brewing or bread making industries. For example, fordairy industry application, a method of the invention is a method forproducing a dairy food, comprising fermenting a culture of lacticacid-producing bacteria (eg, Lactobacillus host cells) for a period oftime to produce lactic acid from the culture, and thereafter inhibitinggrowth of the bacteria by causing expression of crRNA from one or morearrays, systems, vectors, populations or collections of the inventionmixed with the bacteria, whereby lactic acid production by the bacteriais reduced or inhibited. This is useful for reducing food/drink spoilingor undesirable food/drink taste and/or odour. On an example there isincluded an inducible HM-array in the bacteria, wherein the methodcomprises adding an inducer agent after the first period.

References

-   Wall, J. D., B. J. Rapp-Giles, and M. Rousset. 1993.    “Characterization of a small plasmid from Desulfovibrio    desulfuricans and its use for shuttle vector construction”. J.    Bacteriol. 175:4121-4128;-   Seyedirashti S et al; J Gen Microbiol. 1992 July; 138(7): 1393-7,    “Molecular characterization of two bacteriophages isolated from    Desulfovibrio vulgaris NCIMB 8303 (Hildenborough)”;-   Driggers & Schmidt, J. gen. Virol. (1970), 6, 421-427, “Induction of    Defective and Temperate Bacteriophages in Caulobacter”.

Concepts: Altering the Relative Ratio of Sub-Populations of First andSecond Bacteria in a Mixed Population of Bacteria, Eg, in Microbiota

-   1. Use of a host modifying (HM) CRISPR/Cas system for altering the    relative ratio of sub-populations of first and second bacteria in a    mixed population of bacteria, the second bacteria comprising host    cells,    -   for each host cell the system comprising components according        to (i) to (iv):—    -   (i) at least one nucleic acid sequence encoding a Cas nuclease;    -   (ii) a host cell target sequence and an engineered host        modifying (HM) CRISPR array comprising a spacer sequence        (HM-spacer) and repeats encoding a HM-crRNA, the HM-crRNA        comprising a sequence that hybridises to the host cell target        sequence to guide said Cas to the target in the host cell to        modify the target sequence;    -   (iii) an optional tracrRNA sequence or a DNA sequence expressing        a tracrRNA sequence;    -   (iv) wherein said components of the system are split between the        host cell and at least one nucleic acid vector that transforms        the host cell, whereby the HM-crRNA guides Cas to the target to        modify the host CRISPR/Cas system in the host cell; and    -   wherein the target sequence is modified by the Cas whereby the        host cell is killed or host cell growth is reduced.-   2. A host modifying (HM) CRISPR/Cas system for the use of concept 1    for modifying a target nucleotide sequence of a bacterial host cell,    the system comprising components according to (i) to (iv):—    -   (i) at least one nucleic acid sequence encoding a Cas nuclease;    -   (ii) a host cell target sequence and an engineered host        modifying (HM) CRISPR array comprising a spacer sequence        (HM-spacer) and repeats encoding a HM-crRNA, the HM-crRNA        comprising a sequence that is capable of hybridising to the host        target sequence to guide said Cas to the target in the host cell        to modify the target sequence;    -   (iii) an optional tracrRNA sequence or a DNA sequence for        expressing a tracrRNA sequence;    -   (iv) wherein said components of the system are split between the        host cell and at least one nucleic acid vector that can        transform the host cell, whereby the HM-crRNA guides Cas to the        target to modify the host CRISPR/Cas system in the host cell.-   3. The system of concept 2, wherein the vector or vectors lack a Cas    (eg, a Cas9) nuclease-encoding sequence.-   4. The use or system of any preceding concept, wherein each host    cell is of a strain or species found in human microbiota.-   5. The use of concept 1 or 4 for (a) the alteration of the    proportion of Bacteroidetes bacteria in a mixed bacterial    population; (b) reducing the proportion of a Firmicutes    sub-population (host cells) in a mixed bacterial population; (c)    reducing the proportion of a first Firmicutes species (host cells)    in a mixed population, wherein the mixed population comprises a    second Firmicutes species whose growth is not inhibited by said    cRNA; (d) reducing the proportion of a first gram positive bacterial    species (host cells) in a mixed bacterial population, wherein the    mixed population comprises a second gram positive bacterial species    whose growth is not inhibited by said cRNA; (e) reducing the    proportion of a bacterial species (host cells) in a mixed bacterial    population, wherein the mixed population comprises a different    bacterial species whose growth is not inhibited by said cRNA,    wherein the first species has 16s ribosomal RNA-encoding DNA    sequence that is at least 80, 82, 83, 84, 85, 90 or 95% identical to    an 16s ribosomal RNA-encoding DNA sequence of the other species; (f)    reducing the proportion of a first bacterial human gut microbiota    species (host cells, eg, a Firmicutes) in a mixed bacterial    population, wherein the mixed population comprises a different    bacterial species, wherein the different species is a human gut    probiotic species whose growth is not inhibited by said cRNA; or (g)    reducing the proportion of a bacterial human gut microbiota species    ((host cells, eg, a Firmicutes) in a mixed bacterial population,    wherein the mixed population comprises a different bacterial    species, wherein the different species is a human gut commensal    species whose growth is not inhibited by said cRNA.-   6. The system of concept 2 or 3 for (a) the alteration of the    proportion of Bacteroidetes bacteria in a mixed bacterial    population; (b) reducing the proportion of a Firmicutes    sub-population (host cells) in a mixed bacterial population; (c)    reducing the proportion of a first Firmicutes species (host cells)    in a mixed population, wherein the mixed population comprises a    second Firmicutes species whose growth is not inhibited by said    cRNA; (d) reducing the proportion of a first gram positive bacterial    species (host cells) in a mixed bacterial population, wherein the    mixed population comprises a second gram positive bacterial species    whose growth is not inhibited by said cRNA; (e) reducing the    proportion of a bacterial species (host cells) in a mixed bacterial    population, wherein the mixed population comprises a different    bacterial species whose growth is not inhibited by said cRNA,    wherein the first species has 16s ribosomal RNA-encoding DNA    sequence that is at least 80, 82, 83, 84, 85, 90 or 95% identical to    an 16s ribosomal RNA-encoding DNA sequence of the other species; (f)    reducing the proportion of a first bacterial human gut microbiota    species (host cells, eg, a Firmicutes) in a mixed bacterial    population, wherein the mixed population comprises a different    bacterial species, wherein the different species is a human gut    probiotic species whose growth is not inhibited by said cRNA; or (g)    reducing the proportion of a bacterial human gut microbiota species    (host cells, eg, a Firmicutes) in a mixed bacterial population,    wherein the mixed population comprises a different bacterial    species, wherein the different species is a human gut commensal    species whose growth is not inhibited by said cRNA; wherein (a)    to (g) are for treating or preventing in a human or animal    subject (i) a microbiota infection by said bacterial species whose    proportion is reduced; or (ii) a disease or condition mediated by    said bacterial species whose proportion is reduced.-   7. The use or system of concept 5 or 6 for increasing the relative    ratio of Bacteroidetes versus Firmicutes.-   8. The use or system of any preceding concept, wherein said Cas    nuclease is provided by an endogenous Type II CRISPR/Cas system of    the cell.-   9. The use or system of any preceding concept, wherein component (i)    is endogenous to the host cell.-   10. The use or system of any preceding concept, wherein the target    sequence is comprised by an antibiotic resistance gene, virulence    gene or essential gene of the host cell.-   11. The use or system of any preceding concept, wherein the target    sequence is a host chromosomal sequence.-   12. The use or system of any preceding concept, wherein    alternatively HM-crRNA and tracrRNA are comprised by a single guide    RNA (gRNA), eg provided by the vector.-   13. The use or system of any preceding concept, wherein the host    cell comprises a deoxyribonucleic acid strand with a free end    (HM-DNA) encoding a HM-sequence of interest and/or wherein the    system comprises a sequence encoding the HM-DNA, wherein the HM-DNA    comprises a sequence or sequences that are homologous respectively    to a sequence or sequences in or flanking the target sequence for    inserting the HM-DNA into the host genome (eg, into a chromosomal or    episomal site).-   14. An engineered nucleic acid vector for the use of concept 1 for    modifying a bacterial host cell comprising an endogenous CRISPR/Cas    system, the vector    -   (g) comprising nucleic acid sequences for expressing a plurality        of different crRNAs (eg, comprised by gRNAs) for use in a        CRISPR/Cas system or use according to any preceding concept; and    -   (h) optionally lacking a nucleic acid sequence encoding a Cas        nuclease,    -   wherein a first of said crRNAs is capable of hybridising to a        first nucleic acid sequence in said host cell; and a second of        said crRNAs is capable of hybridising to a second nucleic acid        sequence in said host cell, wherein said second sequence is        different from said first sequence; and    -   (i) the first sequence is comprised by an antibiotic resistance        gene (or RNA thereof) and the second sequence is comprised by an        antibiotic resistance gene (or RNA thereof); optionally wherein        the genes are different;    -   (j) the first sequence is comprised by an antibiotic resistance        gene (or RNA thereof) and the second sequence is comprised by an        essential or virulence gene (or RNA thereof);    -   (k) the first sequence is comprised by an essential gene (or RNA        thereof) and the second sequence is comprised by an essential or        virulence gene (or RNA thereof); or    -   (l) the first sequence is comprised by a virulence gene (or RNA        thereof) and the second sequence is comprised by an essential or        virulence gene (or RNA thereof).-   15. The vector of concept 14 inside a host cell comprising one or    more Cas that are operable with cRNA (eg, single guide RNA) encoded    by the vector.-   16. The use, system or vector of any preceding concept, wherein the    HM-CRISPR array comprises multiple copies of the same spacer.-   17. The use, system or vector of any preceding concept, wherein the    vector(s) comprises a plurality of HM-CRISPR arrays.-   18. The use, system or vector of any preceding concept, wherein each    vector is a virus or phage.-   19. The use, system or vector of any preceding concept, wherein the    system or vector comprises two, three or more of copies of nucleic    acid sequences encoding crRNAs (eg, gRNAs), wherein the copies    comprise the same spacer sequence for targeting a host cell sequence    (eg, a virulence, resistance or essential gene sequence).-   20. The use, system or vector of concept 20, wherein the copies are    split between two or more vector CRISPR arrays.-   21. A bacterial host cell comprising a system or vector recited in    any preceding concept.-   22. The use, system, vector or cell of any preceding concept,    wherein the array is in combination with an antibiotic agent; or the    use comprising exposing the host cells to a first antibiotic,    wherein the target sequence is comprised by an antibiotic resistance    gene for resistance to said first antibiotic.-   23. The use, system, vector or cell of any preceding concept,    wherein the host cell is a Staphylococcus, Streptococcus,    Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio, or    Clostridium host cell.-   24. The use, system or cell of any one of concepts 1 to 13 or 16 to    23, wherein each vector is according to concept 14 or 15.-   25. The use, system, vector or cell of any preceding concept wherein    host cell population growth is reduced by of at least 5-fold    compared to the growth of a population of said host cells not    transformed with said HM-array or a nucleotide sequence encoding    said gRNA.-   26. The use, system, vector or cell of any preceding concept wherein    host cell population growth on a surface is inhibited. In an    example, the population is in contact with a human tissue surface    (eg, a gut tissue surface, eg, in vivo or ex vivo.).-   27. The use, system, vector or cell of any preceding concept wherein    the first bacteria are probiotic, commensal or symbiotic with humans    (eg, in the human gut).-   28. The use, system, vector or cell of any preceding concept wherein    the first and second bacteria are both Firmicutes and are bacteria    of different species or strains; or wherein the first bacteria are    Enterobacteriaceae and the second bacteria are Firmicutes.-   29. The use, system, vector or cell of any preceding concept wherein    the host cells are archaeal cells instead of bacterial cells or each    population is an archaeal population instead of a bacterial    population.-   30. The use of any one of concepts 1, 4, 5, 7-13, 16-20 and 22-29    for treating an industrial or an ex vivo medical fluid, surface,    apparatus or container; or for treating a waterway, water, a    beverage, a foodstuff or a cosmetic, wherein the host cell(s) are    comprised by or on the fluid, surface, apparatus, container,    waterway, water, beverage, foodstuff or cosmetic.-   31. The use, system, vector or cell of any preceding concept,    wherein the HM-cRNA or gRNA comprises a sequence that is capable of    hybridising to a host cell target protospacer sequence that is a    adjacent a NNAGAAW or NGGNG protospacer adjacent motif (PAM).-   32. A nucleic acid vector according to, or for use in, the use,    system or cell of any preceding concept, the vector comprising more    than 1.4 kb of exogenous DNA sequence, wherein the exogenous DNA    encodes one or more components of a CRISPR/Cas system and comprises    an engineered array for expressing HM-crRNAs or gRNAs in host cells,    wherein the exogenous sequence is devoid of a nucleotide sequence    encoding a Cas nuclease that is cognate to the cRNA(s) or gRNA(s);    wherein at least 2 different cRNAs or gRNAs are encoded by the    exogenous DNA (eg, by at least 2 HM-CRISPR arrays).-   33. The vector of concept 32, wherein the vector is a viral vector    capable of transforming host cells.-   34. The vector of concept 32 or 33, wherein the cRNAs or gRNAs are    capable of hybridising in host cells to respective target    protospacer sequences, wherein each protospacer sequence is    comprised by an antibiotic resistance or essential host gene.-   35. The vector of any one of concepts 34 to 36, wherein the host    cells are cells of a human microbiota species.

Embodiments Harnessing Wild-Type Endogenous Cas for Population GrowthInhibition & Treatment of Bacteria on Surfaces

1. Use of wild-type endogenous Cas nuclease activity of a bacterial hostcell population to inhibit growth of the population, wherein thepopulation comprises a plurality of host cells and each host cell has anendogenous CRISPR/Cas system having wild-type Cas nuclease activity, theuse comprising transforming host cells of the population, wherein eachtransformed host cell is transformed with an engineered nucleotidesequence for providing host modifying (HM) cRNA or guide RNA (gRNA) inthe host cell, the HM-cRNA or gRNA comprising a sequence that is capableof hybridising to a host cell target protospacer sequence for guidingendogenous Cas to the target, wherein the cRNA or gRNA is cognate to anendogenous Cas nuclease of the host cell that has said wild-typenuclease activity and following said transformation of the host cellsgrowth of the population is inhibited.

The host cells may be of the same species or strain.

2. The use of embodiment 1, wherein the inhibition of host cellpopulation growth is a reduction in growth of at least 5-fold comparedto the growth of a population of said host cells not transformed withsaid engineered nucleotide sequence.3. The use of embodiment 1, wherein population growth on a surface isinhibited.4. The use of embodiment 2, wherein population growth on a surface isinhibited.5. The use of embodiment 1, said inhibiting comprising using aHM-CRISPR/Cas system for killing or reducing the growth of said hostcells, for each host cell the system comprising components according to(i) to (iv):—(i) at least one nucleic acid sequence encoding said Cas nuclease;(ii) an engineered host modifying HM-CRISPR array comprising a spacersequence (HM-spacer) and repeats encoding said HM-crRNA;(iii) an optional tracrRNA sequence or a DNA sequence expressing atracrRNA sequence;(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that transforms the host cell,whereby the HM-crRNA guides said Cas to the target to modify the targetsequence;wherein the target sequence is modified in host cells by the Cas wherebythe host cells are killed or host cell growth is reduced.6. The use of any preceding embodiment, for altering the relative ratioof sub-populations of first and second bacteria in a mixed population ofbacteria, the second bacteria comprising said host cells.7. The use of embodiment 6, wherein the host cells are of a strain orspecies found in human microbiota.8. The use of embodiment 6 or 7, wherein the host cells are mixed withcells of a different strain or species, wherein the different cells areEnterobacteriaceae or bacteria that are probiotic, commensal orsymbiotic with humans (eg, in the human gut).9. The use of any preceding embodiment for the alteration of theproportion of Bacteroidetes bacteria in a mixed bacterial populationcomprising Bacteroidetes bacteria and other bacteria, optionally forincreasing the relative ratio of Bacteroidetes versus one, more or allFirmicutes species (eg, versus Streptococcus) in the population.10. The use of any preceding embodiment for altering the relative ratioof first bacteria versus second bacteria in a mixed population, whereinthe first and second bacteria are both Firmicutes and are bacteria ofdifferent species or strains, the second bacteria comprising host cells.In an example, the use increases the proportion of first to versussecond bacteria.11. The use of embodiment 1, wherein the engineered nucleotide sequenceis not in combination with an exogenous Cas nuclease-encoding sequence.12. The use of embodiment 5, wherein the vector or vectors lack a Casnuclease-encoding sequence.13. The use of embodiment 1, wherein each host cell is of a strain orspecies found in human microbiota.14. The use of embodiment 6, wherein each host cell is of a strain orspecies found in human microbiota.15. The use of embodiment 13, wherein each host cell is mixed with cellsof a different strain or species, wherein the different cells areEnterobacteriaceae or bacteria that are probiotic, commensal orsymbiotic with humans (eg, in the human gut).16. The use of embodiment 1, wherein the use alters the proportion ofBacteroidetes bacteria in a mixed bacterial population comprisingBacteroidetes bacteria and other bacteria, optionally wherein the usealters the relative ratio of Bacteroidetes versus one, more or allFirmicutes (eg, Streptococcus) species in the population.17. The use of embodiment 1, wherein the first and second bacteria areboth Firmicutes and the use alters the relative ratio of the firstversus the second bacteria in the mixed population. In an example, theuse increases the proportion of first to versus second bacteria.18. The use of embodiment 1, wherein said Cas nuclease is provided by ahost cell endogenous Type II CRISPR/Cas system and/or the HM-cRNA orgRNA comprises a sequence that is capable of hybridising to a host celltarget protospacer sequence that is a adjacent a 5′-NNAGAAW-3′protospacer adjacent motif (PAM).19. The use of embodiment 5, wherein said Cas nuclease is provided by ahost cell endogenous Type II CRISPR/Cas system.20. The use of embodiment 5, wherein component (iii) is endogenous tothe host cell.21. The use of embodiment 5, wherein each transformed host cellcomprises an endogenous RNase III that is operable with component (ii)in the production of said HM-crRNA in the cell.22. The use of embodiment 1, wherein the target sequence is comprised byan antibiotic resistance gene, virulence gene or essential gene of thehost cell.23. The use of embodiment 1, wherein the engineered nucleotide sequenceis in combination with an antibiotic agent.24. The use of embodiment 5, wherein the HM-crRNA and tracrRNA arecomprised by a single guide RNA (gRNA).25. The use of embodiment 1, wherein transformed host cells eachcomprise a deoxyribonucleic acid strand with a free end (HM-DNA)encoding a HM-sequence of interest, wherein the HM-DNA comprises asequence or sequences that are homologous respectively to a sequence orsequences in or flanking the target sequence for inserting the HM-DNAinto the host genome, wherein HM-DNA sequences are inserted into hostcell genomes.26. The use of embodiment 1, comprising expressing in host cells aplurality of different crRNAs (or gRNAs) for hybridising to host cellprotospacer target sequences; wherein a first of said crRNAs (or gRNAs)is capable of hybridising to a first protospacer nucleic acid sequence;and a second of said crRNAs (or gRNAs) is capable of hybridising to asecond protospacer nucleic acid sequence, wherein said second sequenceis different from said first sequence; and

-   (a) the first sequence is comprised by an antibiotic resistance gene    (or RNA thereof) and the second sequence is comprised by an    antibiotic resistance gene (or RNA thereof); optionally wherein the    genes are different;-   (b) the first sequence is comprised by an antibiotic resistance gene    (or RNA thereof) and the second sequence is comprised by an    essential or virulence gene (or RNA thereof);-   (c) the first sequence is comprised by an essential gene (or RNA    thereof) and the second sequence is comprised by an essential or    virulence gene (or RNA thereof); or-   (d) the first sequence is comprised by a virulence gene (or RNA    thereof) and the second sequence is comprised by an essential or    virulence gene (or RNA thereof).    27. The use of embodiment 6, wherein the host cells are comprised by    a mixed bacterial population comprised by a human or animal subject    and the use (i) treats in the subject an infection by said host    cells comprised by the mixed population; (ii) treats or reduces the    risk in the subject of a condition or disease mediated by said host    cells; (iii) reduces body odour of the human that is caused or    mediated by said host cells; or (iv) is a personal hygiene treatment    of the human.    28. The use of embodiment 1, wherein the use treats or reduces the    risk of an infection by said host cells in a human or animal    subject, wherein host cells each comprise an antibiotic resistance    gene (for resistance to a first antibiotic) which comprises said    target protospacer sequence, wherein the use comprises administering    the engineered nucleotide sequence and the first antibiotic to the    subject, wherein the infection is reduced or prevented in the    subject.    29. The use of embodiment 1, wherein each engineered nucleotide    sequence further comprises an antibiotic resistance gene, wherein    the HM-crRNA or gRNA does not target the antibiotic resistance gene    and the use comprises exposing the population to said antibiotic and    a plurality of said engineered sequences, thereby promoting    maintenance of HM-crRNA or gRNA-encoding sequences in host cells.    30. The use of embodiment 1, wherein the host cells are gram    positive cells or Streptococcus, Staphylococcus, Pseudomonas,    Salmonella, Listeria, E coli, Desulfovibrio, V cholerae or    Clostridium cells.    31. The use of embodiment 1 for treating an industrial or medical    fluid, surface, apparatus or container; or for treating a waterway,    water, a beverage, a foodstuff or a cosmetic, wherein the host cells    are comprised by or on the fluid, surface, apparatus, container,    waterway, water, beverage, foodstuff or cosmetic, and wherein growth    of the host cell population is inhibited thereby carrying out said    treatment. In an alternative, any embodiment is dependent from any    preceding embodiment.

Aspects: Horizontal Transfer Between Carrier & Host Cells in MixedPopulations

-   1. A method for producing a mixed bacterial population comprising    carrier bacteria, wherein the population comprises first and second    sub-populations of first and second bacteria respectively, wherein    the sub-populations are bacteria of first and second species that    are different from each other and the second bacteria comprise a    plurality of host cells, wherein the carrier bacteria are first    bacteria cells each comprising an engineered nucleotide sequence for    providing host cell modifying (HM) cRNA or guide RNA (gRNA) in the    host cells, the HM-cRNA or gRNA comprising a sequence that is    capable of hybridising to a host cell target protospacer sequence    for guiding a first Cas nuclease to the target to modify the target,    wherein the carrier bacteria do not comprise the target sequence,    the method comprising    -   a. Providing a plurality of nucleic acids, each comprising a        said engineered nucleotide sequence;    -   b. Combining said plurality of nucleic acids with a first mixed        population comprising first and second sub-populations of the        first and second bacterial species respectively, the second        sub-population comprising host cells;    -   c. Allowing the nucleic acids to transform cells of said first        sub-population in the presence of the host cells, thereby        producing a second mixed population comprising said carrier        cells and said host cells, wherein said engineered nucleotide        sequence comprised by carrier cells is capable of horizontal        transfer to host cells to transform host cells for production of        said HM-cRNA or gRNA in transformed host cells.-   2. The method of aspect 1, further comprising obtaining the second    mixed population.-   3. The method of aspect 1, further comprising isolating a plurality    of carrier cells from the second mixed population.-   4. The method of aspect 1, further comprising producing said HM-cRNA    or gRNA in the transformed host cells, wherein said HM-crRNA or gRNA    sequence hybridises to target protospacer sequence in said    transformed host cells and guides the first Cas nuclease to the    target, thereby modifying the target with the first Cas nuclease.-   5. The method of aspect 4, further comprising obtaining host cells    comprising said target modification (eg, wherein the host cells are    comprised by a mixed population comprising said first bacterial    species).-   6. The method of aspect 1, wherein the cRNA or gRNA is cognate to an    endogenous Cas nuclease of the host cells, wherein the nuclease is    said first Cas nuclease.-   7. The method of aspect 1, wherein the cRNA or gRNA is cognate to an    endogenous Cas nuclease of the carrier cells, wherein the nuclease    is said first Cas nuclease.-   8. The method of aspect 7, wherein the nuclease has wild-type    nuclease activity.-   9. The method of aspect 1, 6, 7 or 8, wherein the first Cas nuclease    is a Cas9.-   10. The method of aspect 9, wherein the Cas9 is a Streptococcus    Cas9.-   11. The method of aspect 1, wherein each engineered nucleotide    sequence is comprised by a respective nucleic acid vector, wherein    the vectors are capable of horizontal transfer between the carrier    and host cells.-   12. The method of aspect 1 or 11, wherein each engineered sequence    is comprised by a respective mobile genetic element, eg, a    transposon or plasmid.-   13. The method of aspect 1, wherein following said transformation of    host cells, growth of the host cell sub-population is inhibited.-   14. The method of aspect 13, wherein the inhibition of host cell    population growth is at least 5-fold compared to the growth of a    population of said host cells not transformed with said engineered    nucleotide sequence.-   15. The method of aspect 13 or 14, wherein host cell population    growth on a surface is inhibited.

In an alternative, any aspect is dependent from any preceding aspect.

In an example, the method is a method of treating or preventing adisease or condition in a human, animal or plant subject, eg, asdescribed herein, wherein the method effects said treatment orprevention. The invention provides a mixed bacterial population obtainedor obtainable by the method for such a method of treating or preventing.

In an example, the method is carried out on a mixed bacterial populationof an environment, equipment, apparatus, container, waterway, water,fluid, foodstuff, beverage, microbiota, microbiome or cosmetic, eg, asdescribed herein, wherein the method reduces the proportion of hostcells compared to first cells.

In an example, the product of the method is for administration to thegut of a human or non-human animal for treating or preventing obesity,diabetes or IBD of the human or animal.

In an example, the first and second species are species of human ornon-human animal gut commensal or symbiotic bacteria.

The product of the method is useful as it can be adminstered (eg,intranasally) to a human or animal so that the bacteria populate one ormore microbiomes (eg, gut microbiome) of the human or animal. The firstcells act as carriers, especially when those cells are non-pathogenic tothe human or animal (eg, non-pathogenic in the gut microbiome). Themicrobiome can be any other micribiome or microbiota populationdisclosed herein.

In an example, the first second bacterial species is capable ofpopulating the gut microbiota of a human or non-human animal, and thefirst bacteria are commensal or symbiotic with humans or animals.Usefully, the first bacteria can be safely administered to the human oranimal and can act as a carrier for transfer of engineered sequencesthereafter to host cells of the microbiota.

In an example, the engineered sequence is comprised by any array orvector disclosed herein. In an example, the method uses any CRISPR/Cassystem disclosed herein. In an example the first cell is a Bacteroidetes(eg, Bacteroidales or Bacteroides) cell; Lactobacillus (eg, acidophilus(eg, La-5, La-14 or NCFM), brevis, bulgaricus, plantarum, rhammosus,fermentum, caucasicus, helveticus, lactis, reuteri or casei eg, caseiShirota); Bifidobacterium (eg, bifidum, breve, longum or infantis);Streptococcus thermophiles; Enterococcus faecium; Alistipes;Alkaliflexus; Parabacteroides; Tannerella; E coli; or Xylanibacter cell.

In an example, the host cells are of a human microbiota species and thecarrier cells are cells of a species that is non-pathogenic in saidhuman microbiota, wherein the target sequence is not comprised by thegenome of the carrier cells, the engineered sequence being comprised bya MGE comprising an oriT that is operable in the carrier and host cells,wherein the MGE is capable of horizontal transfer from the carrier cellto the host cell. In an example, the engineered sequence, MGE or vectoris comprised by a bacteriophage, the bacteriophage being capable ofinfecting the first cells (carriers) to introduce the MGE into the first(carrier) cells. Thereafter the MGE is capable of horizontal transfer tohost cells.

In an example, the first cells are Bacteroidetes or Prevotella cells;optionally wherein the MGE is capable of horizontal transfer from thefirst cell species to Firmicutes species (host cells) of said humanmicrobiota. The latter is useful, for example, for treating orpreventing obesity in a human when the target sequence is comprised bythe Firmicutes, but not the first (carrier) cells.

The following numbered paragraphs describe some of the aspects of theinvention. The invention provides, at least:

1. A method of modifying a mixed population of microbiota bacteria, themixed population comprising a first and a second bacterialsub-population of a first and a second microbiota species respectively,wherein the species are different, the second bacterial sub-populationcomprising a host cell population, the method comprising combining themixed population of microbiota bacteria with multiple copies ofengineered nucleic acid sequences encoding host modifying (HM) crRNAs,and expressing HM-crRNAs in host cells, wherein each engineered nucleicacid sequence is operable with a Cas nuclease in a respective host cellto form a HM-CRISPR/Cas system and the engineered sequence comprisesspacer and repeat sequences encoding a HM-crRNA; the HM-crRNA comprisinga sequence that is capable of hybridizing to a host cell target sequenceto guide Cas nuclease to the target sequence in the host cell; andoptionally the HM-system comprises a tracrRNA sequence or a DNA sequenceexpressing a tracrRNA sequence; whereby HM-crRNAs guide Cas modificationof host target sequences in host cells, whereby host cells are killed orthe host cell population growth is reduced, thereby reducing theproportion of said host cell population and altering the relative ratioof said sub-populations of bacteria in the mixed bacterial population.

2. The method of paragraph 1, comprising using endogenous Cas nucleaseof host cells for modification of target nucleotide sequences.

3. The method of paragraphs 1 or 2, comprising reducing host cellpopulation growth by at least 5-fold compared to the growth of a controlpopulation of host cells that have not received said Cas modification.

4. The method of paragraphs 1, 2 or 3, comprising inhibiting host cellpopulation growth on a surface.

5. The method of paragraphs 1, 2, 3, or 4, wherein the first species hasa 16s ribosomal RNA-encoding DNA sequence that is at least 80% identicalto an 16s ribosomal RNA-encoding DNA sequence of the host cell species,wherein the growth of the first bacteria in the mixed population is notinhibited by said HM-system.

6. The method of any of the paragraphs 1-5, wherein the first species isa human gut commensal species and/or a human gut probiotic species.

7. The method of any of the paragraphs 1-6, wherein the first species isa Bacteroidetes (eg, Bacteroides) and optionally the host cells are grampositive bacterial cells.

8. The method of any of the paragraphs 1-7, wherein the host cells areFirmicutes cells.

9. The method of any of the paragraphs 1-8, wherein the host cells areFirmicutes cells.

10. The method of any of the paragraphs 1-9, wherein the host cells areFirmicutes cells.

11. The method of any of the paragraph 1-10, wherein for each host cellthe system comprises components according to (i) to (iv): (i) at leastone nucleic acid sequence encoding a Cas nuclease; (ii) an engineeredHM-CRISPR array comprising a spacer sequence and repeats encoding aHM-crRNA, the HM-crRNA comprising a sequence that hybridises to a hostcell target sequence to guide said Cas to the target in the host cell tomodify the target sequence; (iii) an optional tracrRNA sequence or a DNAsequence expressing a tracrRNA sequence; (iv) wherein said components ofthe system are split between the host cell and at least one nucleic acidvector that transforms the host cell, whereby the HM-crRNA guides Cas tothe target to modify the host target sequence in the host cell; andwherein the target sequence is modified by the Cas whereby the host cellis killed or host cell growth is reduced; the method comprisingintroducing the vectors of (iv) into host cells and expressing saidHM-crRNA in the host cells, allowing HM-cRNA to hybridise to host celltarget sequences to guide Cas to the targets in the host cells to modifytarget sequences, whereby host cells are killed or host cell growth isreduced, thereby altering the relative ratio of said sub-populations inthe mixed population of bacteria.

12. The method of paragraph 11, wherein component (i) is endogenous toeach host cell.

13. The method of paragraph 12, wherein each vector is a virus or phage.

14. The method of paragraph 11, wherein each target sequence is adjacenta NNAGAAW or NGGNG protospacer adjacent motif (PAM).

15. The method of any of the paragraphs 1-14, wherein alternativelyHM-crRNA and tracrRNA are comprised by a single guide RNA (gRNA), themethod comprising introducing said gRNA into host cells or expressingthe gRNA in host cells.

16. The method of any of the paragraphs 1-15 wherein each of the firstand second species is a respective Firmicutes species and the growth ofthe first bacteria is not inhibited by the HM-system.

17. The method of any of the paragraphs 1-16 wherein each of the firstand second species is a respective gram-positive species and the growthof the first bacteria is not inhibited by the HM-system.

18. The method of any one of the paragraphs 1-17 for treating a hostcell infection of a human or animal subject, the method comprisingexposing the host cells to a first antibiotic, wherein target sequencesare each comprised by an antibiotic resistance gene for resistance tosaid first antibiotic, wherein the host cell infection is treated in thesubject.

19. The method of any one of the paragraphs 1-18 for treating orreducing the risk of a disease or condition in a human or animalsubject, wherein the disease or condition is mediated by said secondbacterial species, wherein the first bacteria are probiotic, commensalor symbiotic with humans (eg, in human gut) and wherein the firstbacteria cells do not comprise said target sequence, wherein targetsequence modification by said Cas is carried out and growth of the hostcells is inhibited in said subject but growth of first cells is notinhibited, wherein the disease or condition is treated or risk of thedisease or condition in said subject is reduced.

20. The method of any one of the paragraphs 1-19, for treating anindustrial or medical fluid, surface, apparatus or container; or fortreating a waterway, water, a beverage, a foodstuff or a cosmetic,wherein said host cells are comprised by or on the fluid, surface,apparatus, container, waterway, water, beverage, foodstuff or cosmetic,wherein host cells growth is inhibited, thereby carrying out saidtreatment.

21. The method of any one of the paragraphs 1-20, wherein each host cellis a Staphylococcus, Streptococcus, Pseudomonas, Salmonella, Listeria, Ecoli, Desulfovibrio, Vibrio or Clostridium cell.

22. The method of any one of the paragraphs 1-21, wherein each targetsequence is comprised by an antibiotic resistance gene, virulence geneor essential gene of the host cell.

23. The method of paragraph 7 for increasing the proportion ofBacteroides in the mixed population, wherein said increase is carriedout.

24. The method of paragraph 23, wherein the proportion of Bthetaiotomicron and/or fragalis is increased.

25. The method of paragraph 7, wherein the relative ratio ofBacteroidetes versus Firmicutes or gram-positive host cells comprised bythe mixed population is increased.

26. The method of paragraph 25, wherein the proportion of Bthetaiotomicron and/or fragalis is increased.

27. The method of any of the paragraph 1-24 for favouring commensal orsymbiotic Bacteroidetes in a human or animal.

28. The method of paragraph 27 comprising producing a bacterial culturecomprising the product of claim 1, and administering the culture to ahuman or animal thereby favouring commensal or symbiotic Bacteroidetesin said human or animal.

29. The method of claim 1 for Paneth cell stimulation by gut Bacteroides(e.g., B thetaiotamicron) in a human or animal, wherein the mixedpopulation comprises gut bacteria comprising Bacteroides first bacteriaand the product of claim 1 is produced in said human or animal oradministered to the human or animal, whereby Paneth cells arestimulated.

30. The method of claim 1 for developing an immune response to gutBacteroides (e.g., B fragalis) in a human or animal, wherein the mixedpopulation comprises gut bacteria comprising Bacteroides first bacteriaand the product of claim 1 is produced in said human or animal oradministered to the human or animal, whereby said immune response isdeveloped.

Further Exemplified Clauses of the Invention

The invention also relates to the following Clauses 1 onwards, which areexemplified in worked Examples 6 onwards below, which establish for thefirst time successful host cell targeting in a mixed microbialpopulation of different species and strains using endogenous orexogenous (vector-encoded Cas):—

-   1. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding a Cas nuclease and host modifying        (HM) crRNAs, and    -   b. expressing vector-encoded Cas and HM-crRNAs in host cells,    -    wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with vector-encoded Cas in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas nuclease        to the target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population.-   2. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells,    -    wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;    -    wherein the method reduces host cell population growth by at        least 5, 10-, 100, 1000, 10000, 100000 or 1000000-fold.-   3. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells,    -    wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;    -    wherein the method inhibits host cell population growth on a        surface.-   4. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells,    -    wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;    -    wherein the first species has a 16s ribosomal RNA-encoding DNA        sequence that is at least 80% identical to an 16s ribosomal        RNA-encoding DNA sequence of the host cell species, wherein the        growth of the first bacteria in the mixed population is not        inhibited by said HM-system.-   5. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells,    -    wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;    -    wherein the mixed population of step (a) comprises a third        bacterial species.-   6. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells,    -    wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;    -    wherein the mixed population of step (a) comprises a further        sub-population of bacterial cells of the same species as the        host cells, wherein the bacterial cells of said further        sub-population do not comprise said target sequence. The cells        of said further sub-population are not killed in the presence of        said HM-CRISPR/Cas system or the host cell population growth is        reduced in the presence of said system.-   7. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells, wherein each HM-crRNA is        encoded by a vector engineered nucleic acid sequence and is        operable with a Cas nuclease in a host cell, wherein the        engineered nucleic acid sequence and Cas form a HM-CRISPR/Cas        system and the engineered nucleic acid sequence comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population;    -    wherein each host cell comprises a plurality of said target        sequences.-   8. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells,    -    wherein Cas expression is induced in host cells, whereby said        expressed Cas and HM-crRNAs are combined in the host cells;    -    wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population.-   9. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   b. inducing production of HM-crRNAs in host cells,    -    wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein expression of RNA from the engineered nucleic acid        sequence for production of HM-cRNA is inducible in the host cell        and the engineered sequence and Cas form a HM-CRISPR/Cas system,        the engineered nucleic acid sequence comprising    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population.-   10. A method of modifying a mixed population of microbiota bacteria,    the mixed population comprising a first bacterial sub-population and    a second bacterial sub-population wherein the first sub-population    comprises a first microbiota species and the second sub-population    comprises a host cell population of a second microbiota species,    wherein the second species is a different species than the first    microbiota species, the method comprising    -   a. combining the mixed population of microbiota bacteria with a        plurality of vectors encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells,    -    wherein each HM-crRNA is encoded by a vector engineered nucleic        acid sequence and is operable with a Cas nuclease in a host        cell, wherein the engineered nucleic acid sequence and Cas form        a HM-CRISPR/Cas system and the engineered nucleic acid sequence        comprises    -   (i) a nucleic acid sequence comprising spacer and repeat        sequences encoding said HM-crRNA;    -   (ii) a nucleic acid sequence encoding a sequence of said        HM-crRNA, wherein said HM-crRNA sequence is capable of        hybridizing to a host cell target sequence to guide Cas to the        target sequence in the host cell; and    -    optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -    whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said host cell population and altering the        relative ratio of said sub-populations of bacteria in the mixed        bacterial population.-   11. The method of any one of Clauses 2 to 10, wherein the Cas is    encoded by the vector.-   12. The method of any one of Clauses 2 to 10, wherein the Cas is    encoded by the host cell genome.-   13. The method of any preceding Clause, wherein the method reduces    host cell population growth by at least 5-fold.-   14. The method of any preceding Clause, wherein the method inhibits    host cell population growth on a surface.-   15. The method of any preceding Clause, wherein the first species    has a 16s ribosomal RNA-encoding DNA sequence that is at least 80%    identical to an 16s ribosomal RNA-encoding DNA sequence of the host    cell species, wherein the growth of the first bacteria in the mixed    population is not inhibited by said HM-system.-   16. The method of Clause 15, wherein the first species is a gram    negative species and optionally the second species is a gram    negative species.-   17. The method of any one of Clauses 1 to 15, wherein the first    species is a gram positive species and the second species is a gram    negative species.-   18. The method of any preceding Clause, wherein the mixed population    of step (a) comprises a third species, wherein the third species is    a gram negative species.-   19. The method of any preceding Clause, wherein the mixed population    of step (a) comprises a third bacterial species.-   20. The method of Clause 19, wherein the third species is a gram    positive species.-   21. The method of Clause 20, wherein the first and second species    are gram negative species.-   22. The method of any preceding Clause, wherein the mixed population    of step (a) comprises a further sub-population of bacterial cells of    the same species as the host cells, wherein the bacterial cells of    said further sub-population do not comprise said target sequence.-   23. The method of any preceding Clause, wherein the vectors express    single guide RNAs (HM-gRNAs) comprising HM-crRNA sequences.-   24. The method of any one of Clauses 1 to 22, wherein the method    comprises using endogenous host cell RNase III and/or endogenous    host cell tracrRNA in the production of HM-cRNAs in the host cells.-   25. The method of any preceding Clause, wherein each host cell    comprises a plurality of said target sequences (eg, ribosomal    RNA-encoding sequences, eg, 16s rRNA-encoding sequences).-   26. The method of Clause 25, wherein each host cell comprises at    least 2, 3, 4, 5, 6 or 7 copies of said target sequence.-   27. The method of any preceding Clause, wherein said target sequence    is conserved in bacteria of said second species.    -   For example, the target sequence is conserved in a strain of        bacteria of said second species, and optionally is not conserved        in a second strain of bacterial of said second species.-   28. The method of any preceding Clause, wherein said target sequence    is comprised by an essential gene and/or required for protein    expression in host cells.-   29. The method of any preceding Clause, wherein the hosts cells are    of a first strain of said second species and said genetic target    sequence is present in said strain, but the target sequence is    absent in bacteria of the second species which are of a different    strain.-   30. The method of Clause 29, wherein the mixed population of    step (a) comprises a sub-population of bacteria of said different    strain.    -   For example, the target sequence is conserved in a strain of        bacteria of said second species, and optionally is not conserved        in a second strain of bacterial of said second species.-   31. The method of Clause 30, wherein the first species is a gram    negative species, and the mixed population of step (a) comprises a    sub-population of bacteria of a third species, wherein the third    species is a gram positive species.-   32. The method of Clause 30, wherein the first species is a gram    positive species, and the mixed population of step (a) comprises a    sub-population of bacteria of a third species, wherein the third    species is a gram negative species.-   33. The method of Clause 30, 31 or 32, wherein the second species is    a gram negative species.-   34. The method of any preceding Clause, wherein the second species    is an Enterobacteriaceae species.-   35. The method of any preceding Clause, wherein the second species    is E coli.-   36. The method of any preceding Clause wherein the second species is    a human or animal gut microbiota species.-   37. The method of any preceding Clause wherein each species is an    environmental species, or a human or animal (eg, gut) microbiota    species and/or wherein the host cells are cells of a human    microbiota species.-   38. The method of any preceding Clause, wherein Cas expression is    inducible in host cells.-   39. The method of any preceding Clause, wherein Cas expression is    induced in host cells.-   40. The method of any preceding Clause, wherein HM-cRNA expression    is inducible in host cells.-   41. The method of any preceding Clause, wherein HM-crRNA expression    is induced in host cells.-   42. A vector that is capable of transforming a bacterial host cell,    wherein the vector is capable of accommodating the insertion of (i)    a S pyogenes Cas9 nucleotide sequence that is expressible in the    host cell and (ii) optionally at least one HM-crRNA-encoding    engineered nucleic acid sequence as defined in any preceding Clause,    for use in the method of any preceding Clause, wherein when the    vector comprises (i) (and optionally (ii)) the vector is capable of    transforming the host cell and expressing a Cas (and optionally at    least one HM-crRNA (eg, a gRNA).-   43. The vector of Clause 42, wherein the expressed Cas is a Cas9.

In an alternative, the expressed Cas is a Cas3, eg, an E coli Cas3. Theexpressed Cas is operable with the expressed HM-crRNA(s) in the hostcell to target the Cas to one or more target sequences in the host cell.

-   44. The method or vector of any preceding Clause, wherein said    method is for treating or preventing a disease or condition in a    human or animal; or wherein the method treats or prevents a disease    or condition in a human or animal.-   45. A plurality of bacterial host cells, each comprising a vector of    any one of Clauses 42 to 44, wherein vector-encoded Cas (and    optionally said HMcrRNA(s)) is expressed or expressible in the host    cell, wherein the bacterial cell is comprised by a mixed population    of microbiota bacteria, the mixed population comprising a first    sub-population and a second bacterial sub-population wherein the    first sub-population comprises a first microbiota species and the    second sub-population comprises a host cell population (said    plurality of bacterial host cells) of a second microbiota species,    wherein the second species is a different species than the first    microbiota species.-   46. The plurality of cells of Clause 45 for treating or preventing a    disease or condition in a human or animal.-   47. The method, vector or plurality of cells of any preceding    Clause, wherein the first microbiota species is a human gut    commensal species and/or a human gut probiotic species.-   48. The method, vector or plurality of cells of any preceding    Clause, wherein the first microbiota species is a Bacteroidetes (eg,    Bacteroides) and optionally the host cells are gram positive    bacterial cells.-   49. The method, vector or plurality of cells of any preceding    Clause, wherein the host cell population consists of Firmicutes    cells.-   50. The method, vector or plurality of cells of any preceding    Clause, wherein for each host cell the system comprises components    according to (i) to (iv):—    -   (i) at least one nucleic acid sequence encoding a Cas nuclease;    -   (ii) an engineered HM-CRISPR array comprising a spacer sequence        and repeats encoding a HM-crRNA, the HM-crRNA comprising a        sequence that hybridises to a host cell target sequence to guide        said Cas to the target in the host cell to modify the target        sequence;    -   (iii) an optional tracrRNA sequence or a DNA sequence expressing        a tracrRNA sequence;    -   (iv) wherein said components of the system are split between the        host cell and at least one nucleic acid vector that transforms        the host cell, whereby the HM-crRNA guides Cas to the target to        modify the host target sequence in the host cell; and    -    wherein the target sequence is modified by the Cas whereby the        host cell is killed or host cell growth is reduced;    -    the method comprising introducing the vectors of (iv) into host        cells and expressing said HM-crRNA in the host cells, allowing        HM-cRNA to hybridise to host cell target sequences to guide Cas        to the targets in the host cells to modify target sequences,        whereby host cells are killed or host cell growth is reduced,        thereby altering the relative ratio of said sub-populations in        the mixed population of bacteria.-   51. The method, vector or plurality of cells of any preceding    Clause, wherein each vector is a virus or phage.-   52. The method, vector or plurality of cells of any preceding    Clause, wherein each target sequence is adjacent a NNAGAAW or NGGNG    protospacer adjacent motif (PAM).-   53. The method, vector or plurality of cells of any preceding    Clause, wherein each of the first and second species is a respective    Firmicutes species and the growth of the first bacteria is not    inhibited by the HM-system.-   54. The method, vector or plurality of cells of any preceding    Clause, wherein each of the first and second species is a respective    gram-positive species and the growth of the first bacteria is not    inhibited by the HM-system.-   55. The method, vector or plurality of cells of any preceding Clause    for treating a host cell infection of a human or animal subject, the    method comprising exposing the host cells to a first antibiotic,    wherein target sequences are each comprised by an antibiotic    resistance gene for resistance to said first antibiotic, wherein the    host cell infection is treated in the subject.-   56. The method, vector or plurality of cells of any preceding Clause    for treating or reducing the risk of a disease or condition in a    human or animal subject, wherein the disease or condition is    mediated by said second bacterial species, wherein the first    bacterial species is probiotic, commensal or symbiotic with humans    (eg, in human gut) and wherein the first bacterial species cells do    not comprise said target sequence, wherein target sequence    modification by said Cas is carried out and growth of the host cells    is inhibited in said subject but growth of first bacterial species    cells is not inhibited, wherein the disease or condition is treated    or risk of the disease or condition in said subject is reduced.-   57. The method, vector or plurality of cells of any preceding Clause    for treating an industrial or medical fluid, surface, apparatus or    container; or for treating a waterway, water, a beverage, a    foodstuff or a cosmetic, wherein said host cells are comprised by or    on the fluid, surface, apparatus, container, waterway, water,    beverage, foodstuff or cosmetic, wherein host cells growth is    inhibited, thereby carrying out said treatment.-   58. The method, vector or plurality of cells of any preceding    Clause, wherein each host cell is a Staphylococcus, Streptococcus,    Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio, Vibrio or    Clostridium cell.-   59. The method, vector or plurality of cells of any preceding    Clause, wherein each target sequence is comprised by an antibiotic    resistance gene, virulence gene or essential gene of the host cell.-   60. The method, vector or plurality of cells of any preceding Clause    for increasing the proportion of Bacteroides in the mixed    population, wherein said increase is carried out.-   61. The method, vector or plurality of cells of any preceding    Clause, wherein the proportion of B thetaiotomicron and/or B.    fragilis is increased.-   62. The method, vector or plurality of cells of any preceding    Clause, wherein the relative ratio of Bacteroidetes versus    Firmicutes or gram-positive host cells comprised by the mixed    population is increased.-   63. The method, vector or plurality of cells of any preceding Clause    for favouring commensal or symbiotic Bacteroidetes in a human or    animal.-   64. The method of any preceding Clause, comprising producing a    bacterial culture comprising the product of Clause 1, and    administering the culture to a human or animal thereby favouring    commensal or symbiotic Bacteroidetes in said human or animal.    -   For example the method comprises producing a bacterial culture        comprising the product of claim 1, and administering the culture        to a human or animal thereby favouring commensal or symbiotic        Bacteroidetes in said human or animal.-   65. The method, vector or plurality of cells of any preceding Clause    for Paneth cell stimulation by gut Bacteroides (eg, B    thetaiotamicron) in a human or animal, wherein the first bacterial    species is a Bacteroides species, wherein the method comprises    producing the mixed population comprising said altered ratio in said    human or animal, and administering said mixed population comprising    said altered ratio to the human or animal, whereby Paneth cells are    stimulated.-   66. The method, vector or plurality of cells of any preceding Clause    for developing an immune response to gut Bacteroides (eg, B    fragalis) in a human or animal, wherein the first bacterial species    is a Bacteroides species, wherein the method comprises producing the    mixed population comprising said altered ratio produced in said    human or animal, and administering said mixed population comprising    said altered ratio to the human or animal, whereby said immune    response is developed.

In an embodiment, the plurality of vectors comprises vectors that eachcomprise an nucleotide sequence encoding said Cas for expression of Casin a host cell; and a nucleotide sequence for expressing one or morecrRNAs (eg, comprised by single guide RNAs) in the cell.

In an embodiment, the vectors encode a plurality of Cas proteins (eg, aCas 9 and Cas1 (and/or a Cas2); or Type I CasA, B, C, D, E and Cas3). Inan embodiment, the Cas is a Cas9, dCas9 or a Cas3 (eg, a Type I Cas3 orE coli Cas3 or Salmonella typhimurium Cas3).

In an embodiment, each vector comprises a Cas nuclease-encodingnucleotide sequence and an engineered nucleic acid sequence encodinghost modifying (HM) crRNA.

In an embodiment, the vectors comprise low copy number vectors, eg,plasmids or phagemids. In another embodiment, the vectors comprise highcopy number vectors, eg, plasmids or phagemids.

In an embodiment, each HM-crRNA is encoded by a vector engineerednucleic acid sequence and is operable with vector-encoded Cas in arespective host cell. Multiple HM-crRNAs may be operable withvector-encoded Cas in the same, respective host cell.

In an embodiment, the second species is a gram positive species. In anembodiment, the second species is a gram negative species. In anembodiment, the first or third species is L lactis. In an embodiment,the first or third species is B subtilis. In an embodiment, the first isL lactis and the third species is B subtilis. In an embodiment, thefirst is L lactis and the third species is B subtilis. In an example,the second species is E coli. In an example, the third species iscapable of growth in or on a medium containing PEA, eg, TH mediumcontaining 2.5 g per litre PEA. In an example, the mixed populationcomprises a fourth bacterial sub-population of a fourth bacterialspecies that is different from the first, second and third species.

In an embodiment said vectors are capable of reducing host cellpopulation growth in vitro by at least 5, 10-, 100, 1000, 10000, 100000or 1000000-fold in vitro, eg, on a surface, such as on agar gel. Forexample, the method comprises reducing host cell population growth by atleast said fold compared to the growth of a control population of hostcells that have not received said Cas modification. In an embodimentsaid vectors are capable of reducing host cell population growth invitro by at least 5, 10-, 100, 1000, 10000, 100000 or 1000000-fold invitro in the presence of a third (and optionally also a fourth species),wherein the first, second, third and fourth species are different fromeach other.

In an example, the host cell population is on a surface, whereby hostcell population growth is inhibited on said surface.

In an embodiment, each vector comprises a plurality of HM-CRISPR arrays.In an embodiment, the system or each vector comprises two, three or moreof copies of nucleic acid sequences encoding HM-crRNAs (eg, gRNAs),wherein the copies comprise the same spacer sequence for targeting ahost cell sequence (eg, a virulence, resistance or essential genesequence).

In an example, the method is in vitro. In an example, the method is invivo (eg, in a human or animal, eg, in a gut microbiome thereof, or inor on a plant).

In an example, the method is for (a) the alteration of the proportion ofBacteroidetes bacteria in a mixed bacterial population; (b) reducing theproportion of a Firmicutes sub-population (host cells) in a mixedbacterial population; (c) reducing the proportion of a first Firmicutesspecies (host cells) in a mixed population, wherein the mixed populationcomprises a second Firmicutes species whose growth is not inhibited bysaid cRNA; (d) reducing the proportion of a first gram positivebacterial species (host cells) in a mixed bacterial population, whereinthe mixed population comprises a second gram positive bacterial specieswhose growth is not inhibited by said cRNA; (e) reducing the proportionof a bacterial species (host cells) in a mixed bacterial population,wherein the mixed population comprises a different bacterial specieswhose growth is not inhibited by said cRNA, wherein the first specieshas 16s ribosomal RNA-encoding DNA sequence that is at least 80%identical to an 16s ribosomal RNA-encoding DNA sequence of the otherspecies; (f) reducing the proportion of a first bacterial human gutmicrobiota species (host cells, eg, a Firmicutes) in a mixed bacterialpopulation, wherein the mixed population comprises a different bacterialspecies, wherein the different species is a human gut probiotic specieswhose growth is not inhibited by said cRNA; or (g) reducing theproportion of a bacterial human gut microbiota species ((host cells, eg,a Firmicutes) in a mixed bacterial population, wherein the mixedpopulation comprises a different bacterial species, wherein thedifferent species is a human gut commensal species whose growth is notinhibited by said cRNA, wherein said alteration or reduction is carriedout (eg, ex vivo, in vivo or in a human or animal microbiota (eg, a gutmicrobiota)).

The disease or condition is mediated by, or associated with, thepresence of cells of said second species in the human or animal (eg, ina microbiota or microbiome, such as a gut or skin microbiota ormicrobiome) mentioned herein). In an example, the second species ispathogenic to humans or animals (of the same species of said animal thatis the subject of the method), wherein the method treats or prevents aninfection by cells of said second species.

Any aspect, paragraph, embodiment, example, concept or configurationherein may be combined or applied mutatis mutandis to any Clause 1onwards herein. The method, system, vector(s) or plurality of cells maybe used in or for use in any method or use disclosed herein.

The disclosure of US20160333348 and its continuations andcontinuation-in-part applications (except the present application) isincorporated herein by reference, and any aspect, paragraph, embodiment,example, concept or configuration therein may be combined or appliedmutatis mutandis to any Clause 1 onwards herein.

FURTHER EXAMPLES Example 1: Environmental Treatment or DecontaminationOil, Metal & Mineral Industry

In an embodiment, the host cell is in an a mineral mine or field; in ametal mine or field; in an oil field or in oil or a petrochemical (eg,for any of these when the host is an anaerobic sulphate-reducingbacterium, eg, a Desulfovibrio bacterium). In an example, thiscomposition comprises an oxidising agent (eg, sodium hypochlorite), aquaternary ammonium compound or isothiazolone or is administeredsimultaneously or sequentially with sodium hypochlorite, a quaternaryammonium compound or isothiazolone. An example of a suitable vector foruse in the present invention for modifying a Desulfovibrio bacterialhost is a bacteriophage. The references below describe suitable methodsfor isolating phage that infect Desulfovibrio. For use as a vector inthe present invention, the bacteriophage described by any of thereferences may be used. Alternatively, the vector is provided bynanoparticles.

Heidelberg et al describe the two copies of the nearly identical mu-likebacteriophage DVUO 189-221, DVU2847-79, DVU2688-733 and remnants ofbacteriophage are present in the genome of Desufovibrio vulgarisHildenborough. Such a phage can be a basis on which to design a phagevector for use in the present invention.

References

-   Seyedirashti S et al, J Gen Microbiol. 1991 July; 137(7):1545-9,    “Induction and partial purification of bacteriophages from    Desulfovibrio vulgaris (Hildenborough) and Desulfovibrio    desulfuricans ATCC 13541;-   Seyedirashti S et al, J Gen Microbiol. 1992 July; 138(7): 1393-7,    “Molecular characterization of two bacteriophages isolated from    Desulfovibrio vulgaris NCIMB 8303 (Hildenborough)”;-   *Walker C B et al; Environ Microbiol. 2006 November; 8(11): 1950-9,    “Recovery of temperate Desulfovibrio vulgaris bacteriophage using a    novel host strain”; *[The sequences described in this article have    been deposited in GenBank under Accession No. DQ826728-DQ826732,    incorporated herein by reference]-   Miranda et al, Corrosion Science 48 (2006) 2417-2431, “Biocorrosion    of carbon steel alloys by an hydrogenotrophic sulfate-reducing    bacterium Desulfovibrio capillatus isolated from a Mexican oil field    separator”;-   Eydal et al, The ISME Journal (2009) 3, 1139-1147;    doi:10.1038/ismej.2009.66; published online 11 Jun. 2009,    “Bacteriophage lytic to Desulfovibrio aespoeensis isolated from deep    groundwater”;-   Walker C B et al; Environ Microbiol. 2009 September; 11(9):2244-52.    doi: 10.1111/j.1462-2920.2009.01946.x, “Contribution of mobile    genetic elements to Desulfovibrio vulgaris genome plasticity”.

Example 2: Water or Sewage Treatment or Environmental (Eg, Soil) MetalDecontamination

An alternative application of the invention provides a HM-CRISPR array,HM-CRISPR/Cas system, HM-crRNA, HM-spacer, HM-DNA, HM-Cas orHM-composition as described herein for water or sewage treatment, egwherein the host is a sulphate-reducing bacterium, eg, a Desulfovibriobacterium.

In an example, the target nucleotide sequence in the host is a sequenceof a heavy metal resistance gene. Optionally also the host is aDesulfovibrio bacterium, eg, D vulgaris.

Example 3: Medical Use

An alternative application of the invention provides a HM-CRISPR array,HM-CRISPR/Cas system, HM-crRNA, HM-spacer, HM-DNA, HM-Cas orHM-composition as described herein for treating, preventing or reducing(eg, reducing spread of or expansion of) a bacterial infection in ahuman or animal.

In a first example, the infection is caused by MRSA host cells in ahuman. The host cell is a Staphylococcus aureus host cell and a HM-arrayof the invention is contained in a population of Class I, II or IIIStaphylococcus packaged phage (Caudovirales or Myoviridae phage). Thephage population is administered to a MRSA-infected patient with orwithout methicillin or vancomycin. In one trial, the phage HM-arraystarget (i) the region of 20 nucleotides at the 3′ of the leader promoterof endogenous S aureus CRISPR arrays and (ii) the methicillin resistancegenes in the host cells. When vancomycin is administered, a lower dosethan usual is administered to the patient. It is expected that host cellinfection will be knocked-down and resistance to the phage medicine willnot be established or established at a lower rate or severity thanusual. In other trials, the design is identical except that the phage inthose trials also target the essential S aureus gene ftsZ (Liang et al,Int J Infect Dis. 2015 January; 30:1-6. doi: 10.1016/j.ijid.2014.09.015.Epub 2014 Nov. 5, “Inhibiting the growth of methicillin-resistantStaphylococcus aureus in vitro with antisense peptide nucleic acidconjugates targeting the ftsZ gene”).

A further trial repeated the trials above, but phage K endolysin wasadministered in addition or instead of methicillin.

References

-   1. Jiang W et al, Nucleic Acids Res. 2013 November; 41(20):e188.    doi: 10.1093/nar/gkt780. Epub 2013 Sep. 2, “Demonstration of    CRISPR/Cas9/sgRNA-mediated targeted gene modification in    Arabidopsis, tobacco, sorghum and rice”;-   2. Seed K D et al, Nature. 2013 Feb. 28; 494(7438):489-91. doi:    10.1038/nature 11927, “A bacteriophage encodes its own CRISPR/Cas    adaptive response to evade host innate immunity”;-   3. Semenova E et al, Proc Natl Acad Sci USA. 2011 Jun. 21;    108(25):10098-103. doi: 10.1073/pnas. 1104144108. Epub 2011 Jun. 6,    “Interference by clustered regularly interspaced short palindromic    repeat (CRISPR) RNA is governed by a seed sequence”;-   4. Heler R et al, Mol Microbiol. 2014 July; 93(1): 1-9. doi:    10.1111/mmi.12640. Epub 2014 Jun. 4, “Adapting to new threats: the    generation of memory by CRISPR-Cas immune systems”;-   5. Gomaa A et al, MBio. 2014 Jan. 28; 5(1):e00928-13. doi:    10.1128/mBio.00928-13, “Programmable removal of bacterial strains by    use of genome-targeting CRISPR-Cas systems”;-   6. Fineran P C et al, Proc Natl Acad Sci USA. 2014 Apr. 22;    111(16):E1629-38. doi: 10.1073/pnas.1400071111. Epub 2014 Apr. 7,    “Degenerate target sites mediate rapid primed CRISPR adaptation”;-   7. Wiedenheft et al, Nature. 2011 Sep. 21; 477(7365):486-9. doi:    10.1038/nature 10402, “Structures of the RNA-guided surveillance    complex from a bacterial immune system;-   8. Bondy-Denomy et al, Nature 493, 429-432 (17 Jan. 2013) doi:    10.1038/nature11723, “Bacteriophage genes that inactivate the    CRISPR/Cas bacterial immune system”;-   9. Nuñez J K et al, Nature. 2015 Mar. 12; 519(7542):193-8. doi:    10.1038/nature14237. Epub 2015 Feb. 18, “Integrase-mediated spacer    acquisition during CRISPR-Cas adaptive immunity”.

Example 4: Altering the Ratio of Bacteria in a Mixed Gut MicrobiotaPopulation

Alteration of the ratio of bacteria will be performed according to thepresent example, which is described by reference to knocking-downClostridium dificile bacteria in a mixed gut microbiota sample. Thesample will contain Bacteroides and metronidazole (MTZ)-resistant Cdificile strain 630 sub-populations. Ex vivo the mixed population iscombined with a population of carrier bacteria (Lactobacillusacidophilus La-14 and/or La-5) that have been engineered according tothe invention to contain CRISPR arrays.

Each CRISPR array is comprised on a plasmid that is compatible with thecarrier bacterium and C dificile cells. The array is comprised by aBacteroides thetaiotamicron CTnDot transposon that also comprises oriT,an intDOT sequence, a tetQ-rteA-rteB operon, rteC and the operonxis2c-xis2d-orf3-exc. In one experiment, mob and tra operons areexcluded (instead relying on these supplied by Bacteroides cells towhich the transposons are transferred in the mixture combined with thecarrier bacteria). In another experiment, the mob and tra operons areincluded in the transposons.

Protein translocation across the cytoplasmic membrane is an essentialprocess in all bacteria. The Sec system, comprising at its core anATPase, SecA, and a membrane channel, SecYEG, is responsible for themajority of this protein transport. A second parallel Sec system hasbeen described in a number of Gram-positive species. This accessory Secsystem is characterized by the presence of a second copy of theenergizing ATPase, SecA2; where it has been studied, SecA2 isresponsible for the translocation of a subset of Sec substrates. Incommon with many pathogenic Gram-positive species, Clostridium difficilepossesses two copies of SecA. Export of the S-layer proteins (SLPs) andan additional cell wall protein (CwpV) is dependent on SecA2.Accumulation of the cytoplasmic precursor of the SLPs SlpA and othercell wall proteins is observed in cells expressing dominant-negativesecA1 or secA2 alleles, concomitant with a decrease in the levels ofmature SLPs in the cell wall. Furthermore, expression of eitherdominant-negative allele or antisense RNA knockdown of SecA1 or SecA2dramatically impairs growth, indicating that both Sec systems areessential in C. difficile.

C. difficile Strain 630 (epidemic type X) has a single circularchromosome with 4,290,252 bp (G+C content=29.06%) and a circular plasmidwith 7,881 bp (G+C content=27.9%). The whole genome has been sequencedand found that 11% of the genome consists of mobile genetic elementssuch as conjugative transposons. These elements provide C. difficilewith the genes responsible for its antimicrobial resistance, virulence,host interaction and the production of surface structures. For example,the cdeA gene of C. difficile produces a multidrug efflux pump which wasshown to be homologous to known efflux transporters in the multidrug andtoxic compound extrusion (MATE) family. The protein facilitatesenergy-dependent and sodium-coupled efflux of drugs from cells. Inaddition, the cme gene in C. difficile has been shown to providemultidrug resistance in other bacteria.

The array comprises a R1-S1-R1′ CRISPR unit for targeting a sequence inan essential gene (SecA2) of C dificile cells. In another experiment,targeting is to the cdeA gene in the presence of MTZ and optionally oneor more other anti-C dificile antibiotics. Each spacer (S) comprises a20mer nucleotide sequence of the SecA or cdeA gene, wherein the sequencecomprises a PAM of a C dificile strain 630 CRISPR/Cas system that iscognate to the repeat sequences. Each repeat is identical to a Cdificile strain 630 repeat and has the sequence

(SEQ ID NO: 118) 5′-ATTTACATACCACTTAGTTAATATAAAAC-3′

In an alternative set of experiments, the following sequence is used forthe repeats:

(SEQ ID NO: 119) 5′-GTTTTATATTAACTAAGTGGTATGTAAAT-3′

The repeats function with Cas that is endogenous to the C dificile cellsin the mixed population. The mixed population of bacteria is retrievedas an ex vivo sample from a stool sample of a human patient sufferingfrom C dificile infection. The mixed population is mixed with thecarrier bacteria in vitro and incubated at 37 degrees centigrade underanaerobic conditions to simulate gut conditions in the presence oftetracycline. It is expected that transposons containing the CRISPRarrays will be transferred to Bacteroides and C dificile cells in themixture. Furthermore, it is expected that the target sites in the lattercells will be cut by Cas nuclease action, thus reducing the proportionof C dificile in the mixed population (and increasing the ratio ofBacteroides versus C dificile).

In a follow-on experiment, a drink is produced comprising the carrierbacteria and this is consumed by the human patient once or twice forseveral consecutive days. The patient is also administered withtetracycline during the treatment period. It is expected that stoolanalysis will reveal that the proportion of C dificile in the stoolsamples will reduce (and the ratio of Bacteroides versus C dificile willincrease).

Example 5: Cholera Treatment or Prevention

Reference is made to the World Health Organisation (WHO) Cholera Factsheet No 107 (Updated July 2015). Cholera is an acute diarrhoealinfection caused by ingestion of food or water contaminated with thebacterium Vibrio cholerae. Researchers have estimated that every year,there are roughly 1.4 to 4.3 million cases, and 28 000 to 142 000 deathsper year worldwide due to cholera. The short incubation period of 2hours to 5 days, is a factor that triggers the potentially explosivepattern of outbreaks. Cholera is an extremely virulent disease. Itaffects both children and adults and can kill within hours. About 80% ofpeople infected with V. cholerae do not develop any symptoms, althoughthe bacteria are present in their faeces for 1-10 days after infectionand are shed back into the environment, potentially infecting otherpeople. Among people who develop symptoms, 80% have mild or moderatesymptoms, while around 20% develop acute watery diarrhoea with severedehydration. This can lead to death if left untreated.

Two serogroups of V cholerae—O1 and O139—cause outbreaks. V. cholerae O1causes the majority of outbreaks, while O139—first identified inBangladesh in 1992—is confined to South-East Asia. Non-O1 and non-O139V. cholerae can cause mild diarrhoea but do not generate epidemics.Recently, new variant strains have been detected in several parts ofAsia and Africa. Observations suggest that these strains cause moresevere cholera with higher case fatality rates. The main reservoirs ofV. cholerae are people and water-borne sources such as brackish waterand estuaries, often associated with algal blooms.

Reference is made to Nature. 2013 Feb. 28; 494(7438):489-91. doi:10.1038/nature11927, “A bacteriophage encodes its own CRISPR/Casadaptive response to evade host innate immunity”, Seed K D et al(incorporated herein by reference), which describes that Vibrio choleraeserogroup O1 is the primary causative agent of the severe diarrhoealdisease cholera, and lytic V. cholerae phages have been implicated inimpacting disease burden particularly in the endemic region surroundingthe Bay of Bengal. The authors described the isolation of the ICP1 (forthe International Centre for Diarrhoeal Disease Research, Bangladeshcholera phage 1)-related, V. cholerae O1-specific virulent myovirusesthat are omnipresent amongst cholera patient rice-water stool samplescollected from 2001 to 201114 and in the study described in theirpublication.

The authors explain that ICP1 CRISPR/Cas system consists of two CRISPRloci (designated CR1 and CR2) and six cas genes whose organization andprotein products are most homologous to Cas proteins of the type 1-F(Yersinia pestis) subtype system 17. V. cholerae is divided into twobiotypes, classical and El Tor, the former of which is associated withearlier pandemics and has since been replaced by the El Tor biotype18.The classical strain, V. cholerae 0395, has a CRISPR/Cas systembelonging to the type I-E (Escherichia coli) subtype 17, and to datethere has not been any description of El Tor strains possessing aCRISPR/Cas system. Thus, the origin of the CRISPR/Cas system in ICP1phage is unknown.

The RNA sequence of the CR1 and CR2 consensus direct repeat with thepartially palindromic sequence forming the predicted stem in the crRNAunderlined is as follows:—

GUUAGCAGCCGCAUAGGCUGCUUAAAUA [SEQ ID NO: 75]

In an example of the invention, the or each repeat of the arraycomprises or consists of a sequence that is at least 80, 90, 95, 96, 97,98 or 99% identical to SEQ ID NO: 75 (or is identical to SEQ ID NO: 75).

The majority of spacers in the ICP1 CRISPR show 100% identity tosequences within an 18 kb island found in a subset of V. choleraestrains that include the classical strain 0395 isolated in India in1964, El Tor strain MJ-1236 isolated in Bangladesh in 1994, and severalEl Tor strains collected at the ICDDR, B between 2001-2011. The 18 kbisland resembles the phage inducible chromosomal islands (PICIs) ofGram-positive bacteria, including the prototype Staphylococcus aureuspathogenicity islands (SaPIs). SaPIs are induced to excise, circularizeand replicate following infection by certain phages. They use variedmechanisms to interfere with the phage reproduction cycle to enabletheir own promiscuous spread and this can protect the surroundingbacterial population from further phage predation. The organization ofthe V. cholerae 18 kb island targeted by the ICP1 CRISPR/Cas system issimilar in length, base composition, and organization to that observedin the SaPIs subset of PICIs, with an integrase homologue at one end anda GC content lower than that of the host species (37% compared to47.5%). The 18 kb element is therefore referred to as the V choleraePICI-like element (PLE).

[the nucleotide sequence=SEQ ID NO: 76 (The 32 bp protospacer sequence(SEQ ID NO: 77) is shaded in grey, the present disclosure includes asequence that starts at the first T shaded grey and ends at the last Cshaded grey; and the amino acid sequence=SEQ ID NO: 78]

Seed et al determined that the CR1 and CR2 arrays operate by recognitionof a GA PAM sequence. Seed et al also found that the majority of spacersin the studied ICP1-related phage CRISPR arrays showed identity to V.cholera PLEs. The spacers are shown in the following Table 3; S1 in anarray of the invention is, for example, selected from any one of thesesequences. In an embodiment, S1 is selected from any one of theunderlined sequences.

In an example, the array of the invention (or each array) is anengineered array comprising one, more or all of the underlined spacersequences. The array spacers can comprise a non-naturally occurringarrangement as follows:—

For example, the array comprises a spacer of type 8a and/or 9a, and 0,1, 2, 3, 4, 5 or 6 (but not 7) of types 1a-7a. For example, the arraycomprises a spacer of type 4b, and 0, 1 or 2, 3 (but not 3 or more) of1b-3b. For example, the array comprises a spacer of type 8a and one,more or all of 9a, 4b, 1c, 3d, 1e and 3e. For example, the arraycomprises a spacer of type 9a and one, more or all of 8a, 4b, 1c, 3d, 1eand 3e. For example, the array comprises a spacer of type 4b and one,more or all of 8a, 9a, 1c, 3d, le and 3e. For example, the arraycomprises a spacer of type 1c and one, more or all of 8a, 9a, 4b, 3d, leand 3e. For example, the array comprises a spacer of type 3d and one,more or all of 8a, 9a, 4b, 1c, le and 3e. For example, the arraycomprises a spacer of type 1e and one, more or all of 8a, 9a, 4b, 1c, 3dand 3e. For example, the array comprises a spacer of type 3e and one,more or all of 8a, 9a, 4b, 1c, 3d and 1e.

In another non-naturally occurring arrangement, the vector comprisesfirst and second arrays of the invention, wherein the arrays comprise atleast two spacers selected from 1a to 1g (eg, at least two spacersselected from 8a, 9a, 4b, 1c, 3d, 1e and 3e) wherein the spacers are notspacers of the same ICP1 phage genome, eg, not all spacers ofICP1_2011_A, or ICPI_2006_E, or ICP1_2005_A or ICP1_2004_A (by referenceto the spacers in the table above). Thus, in an embodiment:—

The first array comprises an ICP1_2011_A spacer sequence (eg, 8a and/or9a), and the second array comprises a spacer sequence of ICP1_2006_E,ICPI_2005_A or ICPI_2004 A (eg, one or more spacers selected from 4b,1c, 3d, 1e and 3e).

In an example, the vector comprises 1, 2, 3, 4, 5, 6 or all 7 spacertypes selected from 8a, 9a, 4b, 1c, 3d, 1e and 3e. In an example, thevector comprises multiple copies of one or more of said selected types.In an example, the, some or each array in the vector comprises a firstspacer (nearest the promoter of the array), wherein the first spacer isselected from 8a, 9a, 4b, 1c, 3d, 1e and 3e. Positioning in this way isadvantageous as natural arrays use the first spacer most frequently.

Reference is made to Nucleic Acids Res. 2013 October; 41(19):9033-48.doi: 10.1093/nar/gkt654. Epub 2013 Jul. 30, “High-resolution definitionof the Vibrio cholerae essential gene set with hidden Markov model-basedanalyses of transposon-insertion sequencing data”, Chao M C et al(incorporated by reference), which discloses the coupling ofhigh-density transposon mutagenesis to high-throughput DNA sequencing(transposon-insertion sequencing) enables simultaneous and genome-wideassessment of the contributions of individual loci to bacterial growthand survival. HMM results indicate that 128 genes are required foroptimal growth of V. cholerae in LB. The target sequence of theinvention can be a sequence of any one of these genes (which gene namesand sequences are explicitly incorporated herein by reference for use inproviding target sequences of the vectors of the present invention andfor possible inclusion in the claims herein).

For example, insertion mutants in vc0309 and vc0753, which had averagereads of 5.6 and 4.7, respectively, were severely attenuated in growth.Likewise, vc0237 and vc1773 mutants were less fit than wild-type cellsin an in vitro competition experiment. The list also includes a numberof antitoxin genes from putative toxin/antitoxin addiction loci,including vca0360, vca0477, vca0486 and vca0488. Such genes are presumedto be essential when associated with active toxins.

The authors found the essential V cholerae genes in Table 4. The authorsidentified more than 200 intergenic regions that appear to be essential.

Thus, in an example of the invention when the host cell is a Vibriocholerae cell, the target sequence is a vc0631, vc2024, vc2626,vc2763-vc2767 or vc2768-vc2770 sequence. In an example of the inventionwhen the host cell is a Vibrio cholerae cell, the target sequence is avc0309 and vc0753, vc0237 and vc1773, vca0360, vca0477, vca0486 orvca0488 sequence.

Reference is made to Infect Immun. 2015 September; 83(9):3381-95. doi:10.1128/IAI.00411-15. Epub 2015 Jun. 8, “A Genome-Wide Screen Revealsthat the Vibrio cholerae Phosphoenolpyruvate Phosphotransferase SystemModulates Virulence Gene Expression”, Wang Q et al (incorporated byreference). The authors used a transposon insertion site (TIS)sequencing-based strategy to identify new factors required forexpression of tcpA, which encodes the major subunit of TCP, theorganism's chief intestinal colonization factor. Besides identifyingmost of the genes known to modulate tcpA expression, the screen yieldedptsI and ptsH, which encode the enzyme I (EI) and Hpr components of theV. cholerae phosphoenolpyruvate phosphotransferase system (PTS). Inaddition to reduced expression of TcpA, strains lacking EI, Hpr, or theassociated EIIA(Glc) protein produced less cholera toxin (CT) and had adiminished capacity to colonize the infant mouse intestine. The PTSmodulates virulence gene expression by regulating expression of tcpPHand aphAB, which themselves control expression of toxT, the centralactivator of virulence gene expression.

Thus, in an example of the invention when the host cell is a Vibriocholerae cell, the target sequence is a tcpA sequence or a tcpAmodulator sequence (ie, a nucleotide sequence that modulates tcpA itselfor via its expression product). For example, the sequence is a ptsI orptsH sequence. In an example, the target sequence is sequence of thephosphoenolpyruvate phosphotransferase system (PTS), or a tcpPH, aphABor toxT sequence. In an example the target sequence is a gene sequenceencoding EIIA(Glc) protein.

Suitable target sequences for the present invention are also as shown inTable 5-sequence of any one of the following (Pathogenicity genes areunderlined).

In an embodiment, the cell is a Vibrio (eg, cholera) cell and the targetsequence is a sequence if any of these genes.

Pathogenicity genes are shown in Table 6.

In an embodiment, the cell is a Vibrio (eg, cholera) cell and the targetsequence is a sequence if any of these genes.

Genes from TCP and CTX Pathogenicity Islands

In an embodiment, the cell is a Vibrio (eg, cholera) cell and the targetsequence is an ace, cep, ctxA, ctxB, orfU, zot, rstA, rstB, rstR, acfA,acfB, acfC, tagE, aldA, int, tagA, tagD, tcpA, tcpB, tcpC, tcpD, tcpE,tcpF, tcpH, tcpl, tcpJ, tcpP, tcpQ, tcpR, tcpS, tcpT or toxT sequence.

Example 6: Specific Microbiota Bacterial Population Growth Inhibition ByHarnessing Wild-Type Endogenous Cas

1. Material and Methods

1. 1. Strains

The following strains were used in the course of this Example andExamples 7 and 8: E. coli MG1655, E. coli TOP10, Streptococcusthermophilus LMD-9 (ATCC BAA-491, Manassas, Va.), Streptococcusthermophilus DSM 20617(T) (DSMZ, Braunschweig, Germany), Lactococcuslactis MG1363 and Streptococcus mutans Clarke 1924 DSM 20523 (DSMZ,Braunschweig, Germany).

During the course of media selection and testing of the geneticconstructs different Streptococci strains were used. Streptococcusthermophilus LMD-9 (ATCC BAA-491) and Escherichia coli TOP10 wereconsidered because of their compatible growth requirements. All strainswere cultivated in Todd-Hewitt broth (TH) (T1438 Sigma-Aldrich), inaerobic conditions and at 37° C., unless elsewhere indicated. Thestrains were stored in 25% glycerol at −80° C.

1. 2. Differential Growth Media

All strains were grown on TH media at 37° C. for 20 hours. Selectivemedia for S. thermophilus was TH media supplemented with 3 g 1⁻¹ of2-phenylethanol (PEA). PEA was added to the media and autoclaved at 121°C. for 15 minutes at 15 psi. Agar plates were prepared by adding 1.5%(wt/vol) agar to the corresponding media. When necessary for selectionor plasmid maintenance 30 μg ml⁻¹ kanamycin was used for both S.thermophilus strains and E. coli, and 500 μg ml⁻¹ for S. mutans.

In some cases, depending on the strain and plasmid, a longer incubation,up to 48 hours, may be needed to see growth on media supplemented withPEA. In order to control for the viability of the organisms used, acontrol TH agar must be done in parallel.

1. 3. Cloning

E. coli (One Shot® ThermoFischer TOP10 Chemically Competent cells) wasused in all subcloning procedures. PCR was carried out using Phusionpolymerase. All PCR products were purified with Nucleospin Gel and PCRClean-up by Macherey-Nagel following the manufacturer's protocol. Thepurified fragments were digested with restriction enzyme DpnI in 1×FDbuffer with 1 Cl enzyme in a total volume of 34 μl. The digestedreaction was again purified with Nucleospin Gel and PCR Clean-up byMacherey-Nagel following the manufacturer's protocol. Gibson assemblywas performed in 10 μl reactions following the manufacturer's protocol(NewEngland Biolab).

Plasmid DNA was prepared using Qiagen kits according to themanufacturer's instructions. Modifications for Gram-positive strainsincluded growing bacteria in a medium supplemented with 0.5% glycine andlysozyme to facilitate cell lysis.

1. 4. Transformation

1. 4.1 Electro-Competent E. coli Cells and Transformation

Commercially electrocompetent cells were used for cloning and theexperiments (One Shot® ThermoFischer TOP10 Chemically Competent E.coli). Electroporation was done using standard settings: 1800 V, 25 μFand 200Ω using an Electro Cell Manipulator (BTX Harvard ApparatusECM630). Following the pulse, 1 ml LB-SOC media was added and the cellswere incubated at 37° C. for 1 hour. The transformed cells were platedin LB-agar containing 50 μg ml⁻¹ of kanamycin.

1. 4.2 Preparation of Electro-Competent S. thermophilus Cells

The electroporation protocol was modified from Somkuti and Steinberg,1988. An overnight culture of Streptococcus thermophilus in TH Brothsupplemented with 40 mM DL-threonine (T8375 Sigma-Aldrich) was diluted100-fold in 5 ml of the same media and grown to an OD₆₀₀ between 0.3-0.5(approximately 2.5 hours after inoculation). The cells were collected bycentrifugation at 10,000×g for 10 min at 4° C. and washed three timeswith 5 ml of ice cold wash buffer (0.5 M sucrose+10% glycerol). Afterthe cells were washed, they were suspended to an OD₆₀₀ of 15-30 inelectroporation buffer (0.5 M sucrose, 10% glycerol and 1 mM MgCl₂). Thecells in the electroporation buffer may be kept at 4° C. until use(within one hour) or aliquot 50 μl in eppendorf tubes, freezing them inliquid nitrogen and stored at −80° C. for later use.

1. 4.3 Electroporation S. thermophilus Cells

1 μl of purified plasmid DNA was added to 50 μl of the cell suspensionand electroporation was carried out in 2 mm-gap electroporation cuvettespre-cooled. The electroporation setting were 2500 V, 25 μF and 200Ωusing an Electro Cell Manipulator (BTX Harvard Apparatus ECM630).Immediately after the electric pulse, 1 ml of TH broth was added to thecells and the suspension was kept on ice for 10 minutes, subsequentlythe cells were incubated for 3 h at 37° C. After allowing time forexpression of the resistance gene the cells were plated onto TH-agarplates containing 30 μg ml⁻¹ of kanamycin. Depending on the construct,colonies were visible between 12 and 48 h of incubation at 37° C.

1. 5. Construction of XylS Plasmid

All the plasmids used in this work were based on pBAV1K-T5, which is abroad-host range expression vector derived from the a cryptic plasmidpWV01 from Streptococcus cremoris (Bryksin & Matsumura, 2010), thebackbone was amplified using that contain overhangs for assembly withthe other fragments using Gibson's method.

The xylose inducible system was constructed by cloning the promoter gyrAin front of the XylR repressor (FIG. 1). The XylR repressor wasamplified from Bacillus Subtilis strain SCK6 (Zhang et al. 2011) withthe a reverse primer that includes an overhang for Gibson assembly and aforward primer, that is an ultramer used to introduce the gyrA promoter(Xie et al. 2013) and the corresponding overhang for assembly intopBAV1KT5 backbone. The resulting fragment was flanked by an mCherryamplified from pCL002 (unpublished work) with an ultramer that includePldha+PxylA hybrid promoter (Xie et al. 2013). The three resulting PCRproducts were assembled in a Gibson Master Mix® (NewEngland Biolab)according to manufacturer's instructions. The product was finallytransformed in E. coli TOP10 electrocompetent cells. See FIG. 1.

1. 6. Design and Construction of CRISPR Array Plasmid

Streptococcus thermophilus has 4 distinct CRISPR systems (Sapranauskas,et al. 2011), for this work the type II CRISPR1 (ST1-CRISPR) system waschosen. The design of the target sequence was based on the availablegenome sequence of LMD-9 (GenBank: CP000419.1). The ST1-CRISPR array wasdesigned to contain only the CRISPR array repeats and spacers under axylose inducible promoter (Xie et al. 2013), followed by thecorresponding tracrRNA under a strong constitutive promoter forStreptococci species (Sorg et al. 2014) (FIG. 2, SEQ ID Nos:).

The tracrRNA plays a role in the maturation of crRNA and it is processedby S. thermophilus endogenous RNase III, forming a complex with crRNA.This complex acts as a guide for the endonuclease ST1-Cas9 (Horvath &Barrangou, 2010). After transcription of the synthetic array from thexylose inducible promoter, the endogenous Cas9 and RNAses will processit into a functional gRNA. The gRNA/Cas9 complex will cause a doublestranded break at the target location.

The design of the array used 2 specific target sequences high on GCcontent and a reduced portion of the tracrRNA (ie, a less than completetracrRNA sequence), which has been suggested not to be necessary forproper maturation of crRNA (Horvath & Barrangou, 2010).

The 2 targets were an essential gene (DNA polymerase III subunit alpha)and an antibiotic resistance gene (tetA-like gene) (SEQ ID NOs:).

Primers were used to amplify pBAV1KT5-XylR-PldhA backbone. The CRISPRarray gBlock and the backbone with overhangs were assembled in a GibsonMaster Mix® according to manufacturer's instructions (NewEnglandBiolabs). The product was finally transformed in E. coli TOP10electrocompetent cells.

1. 7. Characterization of Xylose Inducible System in Streptococcusthermophilus LMD-9

Overnight stationary-phase cultures were diluted 1:100 into TH brothwith corresponding antibiotic. Mid-log cells were induced with differentconcentration of D-(+)-xylose (0, 0.001, 0.01, 0.1, 0.5 and 1% wt/vol)and the cell cultures were measured either directly in medium to assessthe extent of autofluorescence of the media, on the cell suspension orthe suspension buffer (PBS buffer). 20 μl samples of the cell cultureswere diluted 1/10 on PBS buffer, on 96-well plates with flat bottoms.Fluorescence of cell suspensions or media was read on a plate reader.mCherry fluorescence was measured using an excitation wavelength of 558nm and emission at 612 nm. Absorbance of the resuspended cells wasmeasured at OD 600 nm. A minimum of three independent biologicalreplicates was done for each experiment.

1.8. Activation of CRISPR Array in S. thermophilus

S. thermophilus LMD-9 and E. coli TOP10 both with the plasmid containingthe CRISPR array targeting the DNA polymerase III and tetA of S.thermophilus were grown overnight in 3 ml cultures supplemented with 30μg ml⁻¹ of kanamycin for plasmid maintenance. The next day 96 well deepwell plates were inoculated with 500 μl of 1/100 of overnight culture infresh TH media, supplemented with 30 g ml⁻¹ kanamycin. Mid-log cellcultures were induced with 1% xylose. The killing effect was tested onS. thermophilus and E. coli alone. For each strain and condition testeda negative control was kept without xylose. The cells were grown till˜OD 0.5 and next 10-fold serially diluted in TH media and using a96-well replicator (Mettler Toledo Liquidator™ 96) 5 μL volume dropswere spotted on TH agar and TH agar supplemented with g 1⁻¹ PEA plates.The plates were incubated for 24H at 37° C. and the colony forming units(CFU) were calculated from triplicate measurements.

2. Results

2.1 Growth Condition and Selective Media

We first set out to establish the bacterial strains and cultivationprotocol that would support growth for all strains we planned to use forthe co-cultivation experiments. We used S. thermophilus strain LMD-9which was able to support a similar growth as E. coli in TH broth at 37°C. (FIG. 3).

Distinguishing the different bacteria from a mixed culture is importantin order to determine cell number of the different species. WithMacConkey agar is possible to selectively grow E. coli, however there isno specific media for selective growth of S. thermophilus. PEA agar is aselective medium that is used for the isolation of gram-positive (S.thermophilus) from gram-negative (E. coli). Additionally, we found thatdifferent concentrations of PEA partially inhibit the growth of othergram positives, which allow for selection between the othergram-positive bacteria used in this work (FIG. 4). 3 g 1⁻¹ of PEA provedto selectively grow S. thermophilus LMD-9 while limiting growth of E.coli.

2.2 Design and Validation of Inducible System

An induction system for Streptococcus species was previously developedbased on the Bacillus megaterium xylose operon (FIG. 5) by creating aheterologous xylose induction cassette (Xyl-S). The xylR and xylApromoters were replaced with S. mutans' constitutively expressed gyrAand ldh promoters respectively. This expression cassette forStreptococcus species showed differences in sensitivity and expressionlevels between different species, however the system was not tested inS. thermophilus (Xie et al. 2013). Therefore we first set out tovalidate the xylose induction cassette in S. thermophilus.

An alternative version of the induction cassette was constructed by onlyreplacing the xylR promoter with the S. mutans' gyrA promoter but leftthe endogenous B. megaterium xylA promoter intact. During the design ofthe xylose inducible system we considered both versions of the induciblepromoter, the natural P_(XylA) promoter found in Bacillus megaterium anda hybrid promoter of the highly conserved promoter P_(ldha) fused withthe repressor binding sites of P_(XylA) promoter (FIG. 5). Only a fewStreptococcus species have been reported to metabolize xylose, and thusthe presence of a regulatory machinery to recognize the xylA promoter inthe other Streptococcus species is not likely. Therefore we constructedboth xylose induction systems but only tested the inducibility ofmCherry with the P_(ldha+XylA) system.

In order to determine mCherry inducible expression by xylose, mid-logcultures of cells with the plasmid (pBAV1KT5-XylR-mCherry-Pldha+XylA)were induced with different concentrations of xylose. Six hours afterthe induction we measured mCherry fluorescence in the cultures, where weobserved substantially higher overall expression levels in cellscarrying the plasmid (FIG. 6). It is worth noticing that the systemshowed a substantial level of basal expression even in the cultureswhere xylose was not added. This means that the system is ‘leaky’ and incontext of the kill-array this can lead to cell death even before thesystem is induced with xylose. However, in the subsequent course of thisstudy we used both versions of the plasmid(pBAV1KT5-XylR-mCherry-P_(ldha+XylA) and pBAV1KT5-XylR-mCherry-PxylA).

2. 3 Design of CRISPR CAS9 Array

In order to determine if the genomic targeting spacers in a CRISPR arraycan cause death in S. thermophilus LMD-9, we inserted the CRISPR arraywe designed into the two xylose inducible systems previously constructed(pBAV1KT5-XylR-mCherry-P_(ldha+XylA) andpBAV1KT5-XylR-mCherry-P_(xylA)). In these plasmids we replaced mCherrywith the gBlock containing the CRISPR array (FIG. 7). The variant withthe P_(ldha+XylA) promoter was expected to be stronger and have a higherbasal activity than the P_(xy1A) (Xie et al. 2013).

2. 4 Inhibition of Bacterial Population Growth Using Endogenous Cas9

After we constructed the plasmids in E. coli, we transformed theplasmids into S. thermophilus. This would allow us to determine if wecould cause cell death of a specific bacterial species. Interestingly,bacterial host population size (indicated by growing bacteria andcounting colony numbers on agar plates) in S. thermophilus exposed tothe plasmid containing the strong P_(ldh+XylA) hybrid promoter was10-fold less when compared to S. thermophilus exposed to the plasmidcontaining the weak, normal P_(xylA) promoter (FIG. 8; 52 colonies withthe strong array expression versus 556 colonies with weak arrayexpression, 10.7-fold difference), the 2 strains having been transformedin parallel using the same batch of electrocompetent S. thermophiluscells. This suggests to us that the plasmid carrying the CRISPR arraytargeting S. thermophilus genes is able to kill the cells using theendogenous Cas nuclease and RNase III, thereby inhibiting populationgrowth by 10-fold.

We expect that weak array expression in host cells transformed by theplasmid comprising the P_(xy1A) promoter led to a degree of cellkilling, albeit much less than with the strong promoter plasmid. Weexpect that population growth inhibition that is greater than theobserved 10-fold inhibition would be determined if a comparison of theactivity of strong array expression was made with S thermophilus that isnot exposed to any array-encoding plasmid (such as bacteria directlyisolated from gut microbiota). Thus, we believe that array (or singleguide RNA) expression in host cells for harnessing endogenous Casnuclease will be useful for providing effective growth inhibition oftarget host cells in environmental, medical and other settings mentionedherein. Co-administration of antibiotic may also be useful to enhancethe growth inhibition, particularly when one or more antibioticresistance genes are targeted.

3. Discussion and Outlook

In this study we set out to design a CRISPR-array to specifically killS. thermophilus using the endogenous Cas9 system. In order to gaincontrol over the killing signal we sought to apply an inducible systemthat can be applied in S. thermophilus. The xylose inducible XylR systemfrom B. megaterium was previously applied in S. mutans (Xie, 2013) butnot in S. thermophilus. In this study we demonstrated the functionalityof the xylR induction system using the designed XylR-mCherry-Pldhacircuit in S. thermophilus. We found 0.1% wt/vol is sufficient to fullyinduce the XylR system in S. thermophilus (FIG. 6).

In order to observe abundance when co-culturing S. thermophilus and E.coli we established that supplementation of the culture media with 3 g1⁻¹ of PEA, allows for the selective growth of S. thermophilus whilelimiting the growth of E. coli (FIG. 4).

A ST1-CRISPR array, targeting the DNA polymerase III subunit alpha and atetA like gene in the S. thermophilus LMD-9 genome, was placed under thexylose inducible promoter (Xie et al. 2013). Targeting these regionsshould lead to a double strand break and thus limit S. thermophilusviability (FIG. 9). Since the engineered array was designed to target S.thermophilus genome using the endogenous CRISPR/Cas machinery to processthe encoded CRISPR array, the array is expected to have no influence ongrowth of unrelated strains such as E. coli, even similar targets couldbe found on its genome. This was successfully tested in a mixedbacterial population (simulating aspects of a human microbiota) asdiscussed in Example 8.

The demonstration of the invention's ability to inhibit host cell growthon a surface is important and desirable in embodiments where theinvention is for treating or preventing diseases or conditions mediatedor caused by microbiota as disclosed herein in a human or animalsubject. Such microbiota are typically in contact with tissue of thesubject (eg, gut, oral cavity, lung, armpit, ocular, vaginal, anal, ear,nose or throat tissue) and thus we believe that the demonstration ofactivity to inhibit growth of a microbiota bacterial species(exemplified by Streptococcus) on a surface supports this utility.

Example 7: Specific Microbiota Bacterial Population Growth Inhibition inDifferent Strains

Example 6 demonstrated specific growth inhibition of Streptococcusthermophilus LMD-9. Here we demonstrate growth inhibition can also beobtained in a second strain: Streptococcus thermophilus DSM 20617.Methods described in Example 6 were, therefore, applied to the latterstrain (except that selective media for S. thermophilus DSM 20617 was THmedia supplemented with 2.5 g 1⁻¹′ of 2-phenylethanol (PEA)).

Streptococcus thermophilus DSM 20617 transformed with the CRISPR arrayplasmids were incubated for recovery in liquid media for a period of 3hours at 37° C. that would allow for expression of kanamycin resistance.After a recovery period, cells were plated in different selection mediain presence of 1% xylose in order to induce cell death, and withoutxylose as a control (FIG. 10). It is evident that; (1) by xyloseinduction the growth of S. thermophilus can be inhibited (around 10-foldfor the ‘strong’ promoter plasmid versus control), (2) the ‘strong’system (pBAV1KT5-XylR-CRISPR-P_(ldhA)) results in more growth reductionthan the ‘weak’ system (pBAV1KT5-XylR-CRISPR-P_(xlA)).

Example 8: Selective Bacterial Population Growth Inhibition in a MixedConsortium of Different Microbiota Species

We next demonstrated selective growth inhibition of a specific bacterialspecies in a mixed population of three species. We selected speciesfound in gut microbiota of humans and animals (S thermophilus DSM20617(T), Lactobacillus lactis and E coli). We included twogram-positive species (the S thermophilus and L lactis) to see if thiswould affect the ability for selective killing of the former species;furthermore to increase difficulty (and to more closely simulatesituations in microbiota) L lactis was chosen as this is aphylogenetically-related species to S thermophilus (as indicated by high16s ribosomal RNA sequence identity between the two species). The Sthermophilus and L lactis are both Firmicutes. Furthermore, to simulatemicrobiota, a human commensal gut species (E coli) was included.

1. Materials & Methods

Methods as set out in Example 6 were used strain (except that selectivemedia was TH media supplemented with 2.5 g 1⁻¹ of 2-phenylethanol(PEA)).

1.1 Preparation of Electro-Competent L. lactis Cells

Overnight cultures of L. lactis in TH media supplemented with 0.5 Msucrose and 1% glycine were diluted 100-fold in 5 ml of the same mediaand grown at 30° C. to an OD₆₀₀ between 0.2-0.7 (approximately 2 hoursafter inoculation). The cells were collected at 7000×g for 5 min at 4°C. and washed three times with 5 ml of ice cold wash buffer (0.5 Msucrose+10% glycerol). After the cells were washed, they were suspendedto an OD₆₀₀ of 15-30 in electroporation buffer (0.5 M sucrose, 10%glycerol and 1 mM MgCl₂). The cells in the electroporation buffer werekept at 4° C. until use (within one hour) or aliquot 50 μl in eppendorftubes, freezing them in liquid nitrogen and stored at −80° C. for lateruse.

Electroporation conditions for all species were as described in Example6.

1.2 Activation of CRISPR Array: Consortium Experiments.

S. thermophilus DSM 20617, L. lactis MG1363 and E. coli TOP10 weregenetically transformed with the plasmid containing the CRISPR arraytargeting the DNA polymerase III and tetA of S. thermophilus. Aftertransformation all cells were grown alone and in co-culture for 3 hoursat 37° C. allowing for recovery to develop the antibiotic resistanceencoded in the plasmid. We decided to use transformation efficiency as aread out of CRISPR-encoded growth inhibition. Therefore, after allowingthe cells for recovery the cultures were plated in TH media, THsupplemented with PEA and MacConkey agar all supplemented withKanamycin, and induced by 1% xylose.

2. Results

2.0 Phylogenetic Distance Between L. lactis, E. Coli and S. thermophilus

The calculated sequence similarity in the 16S rrNA-encoding DNA sequenceof the S. thermophilus and L. lactis was determined as 83.3%. Thefollowing 16S sequences were used: E. coli: AB030918.1, S. thermophilus:AY188354.1, L. lactis: AB030918. The sequences were aligned with needle(http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html ) with thefollowing parameters: -gapopen 10.0-gapextend 0.5-endopen 10.0-endextend0.5-aformat3 pair-snucleotide1-snucleotide2. FIG. 11 shows themaximum-likelihood phylogenetic tree of 16S sequences from S.thermophilus, L. lactis and E. coli.

2.1 Growth Condition and Selective Media

S. thermophilus and L. lactis are commonly used in combination in manyfermented foods and yoghurt. We chose these strains since they arecommonly known to be gut microbes that form an intimate association withthe host and previous characterizations of the 16S ribosomal RNA regionof S. thermophilus and L. lactis have shown that these organisms arephylogenetically closely related (Ludwig et al., 1995). In parallel wealso evaluated the growth of E. coli for our mixed population co-cultureexperiments, since this organism is also commonly found in gut microbecommunities. We first set out to establish the bacterial strains andcultivation protocol that would support growth for all strains weplanned to use for the co-cultivation experiments. We found that allstrains were able to support growth in TH broth at 37° C. (FIG. 3).

Distinguishing the different bacteria from a mixed culture is importantin order to determine cell number of the different species. WithMacConkey agar is possible to selectively grow E. coli, however there isno specific media for selective growth of S. thermophilus. PEA agar is aselective medium that is used for the isolation of gram-positive (S.thermophilus) from gram-negative (E. coli). Additionally, differentconcentrations of PEA partially inhibit the growth of the differentgrams positive species and strains, which allow for selection betweenthe other gram-positive bacteria used in this work. Using 2.5 g 1⁻¹ ofPEA proved to selectively grow S. thermophilus while limiting growth ofL. lactis and E. coli.

All strains were transformed with a plasmid that used the vectorbackbone of pBAV1KT5 that has a kanamycin selection marker; we foundthat using media supplemented with 30 ug ml⁻¹ of kanamycin was enough togrow the cells while keeping the plasmid.

2. 3 Transformation & Selective Growth Inhibition in a Mixed Population

We transformed S. thermophilus, L. lactis and E. coli with plasmidcontaining the CRISPR array and cultured them in a consortium of all thebacterial species combined in equal parts, which would allow us todetermine if we could cause cell death specifically in S. thermophilus.We transformed all the species with either thepBAV1KT5-XylR-CRISPR-P_(XylA) or pBAV1KT5-XylR-CRISPR-P_(ldha+XylA)plasmid.

FIG. 12 shows the selective S thermophilus growth inhibition in aco-culture of E. coli, L. lactis and S. thermophiles harboring eitherthe pBAV1KT5-XylR-CRISPR-P_(xylA) or thepBAV1KT5-XylR-CRISPR-P_(ldhA+XylA) plasmid. No growth difference isobserved between E. coli harboring the pBAV1KT5-XylR-CRISPR-P_(XylA) orthe pBAV1KT5-XylR-CRISPR-P_(ldhA+XylA) plasmid (middle column). However,S. thermophiles (selectively grown on TH agar supplemented with 2.5 gl⁻¹PEA, last column) shows a decrease in transformation efficiency betweenthe pBAV1KT5-XylR-CRISPR-P_(xylA) (strong) or thepBAV1KT5-XylR-CRISPR-P_(ldhA+XylA) (weak) plasmid as we expected. Wethus demonstrated a selective growth inhibition of the target Sthermophilus sub-population in the mixed population of cells.

References

-   Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Patrick    Boyaval, Moineau, S., . . . Horvath, P. (2007). CRISPR Provides    Acquired Resistance Against Viruses in Prokaryotes. Science, 315    (March), 1709-1712.-   Bryksin, A. V, & Matsumura, I. (2010). Rational design of a plasmid    origin that replicates efficiently in both gram-positive and    gram-negative bacteria. PloS One, 5(10), e13244.-   Chan C T Y, Lee J W, Cameron D E, Bashor C J, Collins J J: “Deadman”    and “Passcode” microbial kill switches for bacterial containment.    Nat Chem Biol 2015, 12 (December): 1-7.-   Horvath, P., Romero, D. A., Cofite-Monvoisin, A.-C., Richards, M.,    Deveau, H., Moineau, S., Barrangou, R. (2008). Diversity, activity,    and evolution of CRISPR loci in Streptococcus thermophilus. Journal    of Bacteriology, 190(4), 1401-12.-   Ludwig, E. S., Klipper, R., Magrum L., Wose C., & Stackebrandt, E.    (1985). The phylogenetic position of Streptococcus and Enterococcus.    Journul of Gencwl Microhiologj., 131, 543-55 1.-   Mercenier, A. (1990). Molecular genetics of Streptococcus    thermophilus. FEMS Microbiology Letters, 87(1-2), 61-77. \-   Samar{hacek over (z)}ija, D., Antunac, N., & Havranek, J. (2001).    Taxonomy, physiology and growth of Lactococcus lactis: a review.    Mljekarstvo, 51(1), 35-48. Retrieved from-   Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R., Horvath,    P., & Siksnys, V. (2011). The Streptococcus thermophilus CRISPR/Cas    system provides immunity in Escherichia coli. Nucleic Acids    Research, 39(21), 9275-9282.-   Somkuti, G. A., & Steinberg, D. H. (1988). Genetic transformation of    Streptococcus thermophilus by electroporation. Biochimie, 70(4),    579-585-   Sorg, R. A., Kuipers, O. P., & Veening, J.-W. (2014). Gene    expression platform for synthetic biology in the human pathogen    Streptococcus pneumoniae. ACS Synthetic Biology, 4(3), 228-239.-   Suvorov, a. (1988). Transformation of group A streptococci by    electroporation. FEMS Microbiology Letters, 56(1), 95-99.-   Xie, Z., Qi, F., & Merritt, J. (2013). Development of a tunable    wide-range gene induction system useful for the study of    streptococcal toxin-antitoxin systems. Applied and Environmental    Microbiology, 79(20), 6375-84.-   Zhang, X. Z., & Zhang, Y. H. P. (2011). Simple, fast and    high-efficiency transformation system for directed evolution of    cellulase in Bacillus subtilis. Microbial Biotechnology, 4(1),    98-105.

Example 9: Vector-Encoded System for Selective Species & Strain GrowthInhibition in a Mixed Bacterial Consortium

In Example 8 we surprisingly established the possibility of harnessingendogenous Cas nuclease activity in host bacteria for selectivepopulation growth inhibition in a mixed consortium of different species.We next explored the possibility of instead using vector-encoded Casactivity for selective population growth inhibition in a mixedconsortium of different species. We demonstrated selective growthinhibition of a specific bacterial species in a mixed population ofthree different species, and further including a strain alternative tothe target bacteria. We could surprisingly show selective growthinhibition of just the target strain of the predetermined targetspecies. Furthermore, the alternative strain was not targeted by thevector-encoded CRISPR/Cas system, which was desirable for establishingthe fine specificity of such vector-borne systems in a mixed bacterialconsortium that mimicked human or animal gut microbiota elements.

We selected species found in gut microbiota of humans and animals(Bacillus subtilis, Lactobacillus lactis and E coli). We included twostrains of the human commensal gut species, E coli. We thought it ofinterest to see if we could distinguish between closely related strainsthat nevertheless had sequence differences that we could use to targetkilling in one strain, but not the other. This was of interest as somestrains of E coli in microbiota are desirable, whereas others may beundesirable (eg, pathogenic to humans or animals) and thus could betargets for Cas modification to knock-down that strain.

1. Material and methods1.1. Plasmids and strains

See Tables 7 and 8. All strains were cultivated in Todd-Hewitt broth(TH) (T1438 Sigma-Aldrich), in aerobic conditions and at 37° C., unlesselsewhere indicated. The strains were stored in 25% glycerol at −80° C.

The self-targeting sgRNA-Cas9 complex was tightly regulated by atheophylline riboswitch and the AraC/P_(BAD) expression systemrespectively. Tight regulation of Cas9 is desired in order to be carriedstably in E. coli. The plasmid contained the exogenous Cas9 fromStreptococcus pyogenes with a single guide RNA (sgRNA) targeting E.coli's K-12 strains. Therefore K-12 derived strains TOP10 wassusceptible to double strand self-cleavage and consequent death when thesystem was activated. E. coli strains like Nissle don't have the sametarget sequence therefore they were unaffected by the sgRNA-Cas9activity. See Tables 9-11 below, which show sequences used in Example 9.We chose a target sequence (ribosomal RNA-encoding sequence) that isconserved in the target cells and present in multiple copies (7 copies),which increased the chances of cutting host cell genomes in multipleplaces to promote killing using a single gRNA design.

FIG. 13 shows regulators controlling the expression of spCas9 and theself-targeting sgRNA targeting the ribosomal RNA subunit 16s.

1. 2. Differential growth media

All strains were grown on TH media at 37° C. for 20 hours. Selectivemedia for B. subtilis was TH media supplemented with 2.5 g 1⁻¹ of2-phenylethanol (PEA). PEA was added to the media and autoclaved at 121°C. for 15 minutes at 15 psi. Agar plates were prepared by adding 1.5%(wt/vol) agar to the corresponding media.

1. 3. Cloning

E. coli (One Shot® ThermoFischer TOP10 Chemically Competent cells) wasused in all subcloning procedures. PCR was carried out using Phusion™polymerase. All PCR products were purified with Nucleospin™ Gel and PCRClean-up by Macherey-Nagel™ following the manufacturer's protocol. Thepurified fragments were digested with restriction enzyme DpnI in 1×FDbuffer with 1 Cl enzyme in a total volume of 34 μl. The digestedreaction was again purified with Nucleospin Gel and PCR Clean-up byMacherey-Nagel following the manufacturer's protocol. Gibson assemblywas performed in 10 μl reactions following the manufacturer's protocol(NewEngland Biolab).

Plasmid DNA was prepared using Qiagen kits according to themanufacturer's instructions. Modifications for Gram-positive strainsincluded growing bacteria in a medium supplemented with 0.5% glycine andlysozyme to facilitate cell lysis.

1. 4. Transformation

1. 4.1 Electro-Competent E. coli Cells and Transformation

Commercially electrocompetent cells were used for cloning and theexperiments (One Shot® ThermoFischer TOP10 electrompetent E. coli).Electroporation was done using standard settings: 1800 V, 25 μF and 200Ωusing an Electro Cell Manipulator (BTX Harvard Apparatus ECM630).Following the pulse, 1 ml LB-SOC media was added and the cells wereincubated at 37° C. for 1 hour. The transformed cells were plated inLB-agar containing the corresponding antibiotics.

1.5. Activation of sgRNA-Cas9 in E. coli and Consortium Experiments.

E. coli TOP10 and Nissle both with the plasmid containing the sgRNAtargeting the ribosomal RNA-encoding sequence of K-12 derived strainsand the other bacteria were grown overnight in 3 ml of TH broth. Thenext day the cells were diluted to ˜OD 0.5 and next 10-fold seriallydiluted in TH media and using a 96-well replicator (Mettler ToledoLiquidator™ 96) 4 μL volume drops were spotted on TH agar, TH agar withinducers (1% arabinose and 2 mM theophylline), TH agar supplemented with2.5 g 1⁻¹ PEA and MacConkey agar supplemented with 1% maltose. Theplates were incubated for 20 h at 37° C. and the colony forming units(CFU) were calculated from triplicate measurements.

2. Results

2.1 Specific Targeting of E. coli Strains Using an Exogenous CRISPR-Cas9System

We first tested if the system could differentiate between two E. colistrains by introducing the killing system in both E. coli TOP10 andNissle.

2.1 Targeting of E. coli using an exogenous CRISPR-Cas9 system in amixed culture

Serial dilutions of overnight cultures were done in duplicate for bothE. coli strains, B. subtilis, L. lactis, and in triplicate for the mixedcultures. All strains were grown at 37° C. for 20 hours in selectiveplates with and without the inducers. Induction of the system activatesthe sgRNA-Cas9 targeting K-12 derived strains, while leaving intact theother bacteria.

Distinguishing the different bacteria from a mixed culture is importantin order to determine cell numbers of the different species anddetermine the specific removal of a species. MacConkey agar selectivelygrows E. coli, PEA agar is a selective medium that is used for theisolation of gram-positive (B. subtilis) from gram-negative (E. coli).Additionally, we found that different concentrations of PEA partiallyinhibit the growth of other gram positives. 2.5 g 1⁻¹ of PEA proved toselectively grow B. subtilis while limiting growth of E. coli and L.lactis.

FIG. 14 shows specific targeting of E. coli strain by the inducible,exogenous, vector-borne CRISPR-Cas system. The sgRNA target the genomeof K-12 derived E. coli strain E. coli TOP10, while the other E. colistrain tested was unaffected.

FIG. 15 shows spot assay with serial dilutions of individual bacterialspecies used in this study and mixed culture in TH agar withoutinduction of the CRISPR-Cas9 system.

FIG. 16 shows a spot assay of the dilution 10³ on different selectivemedia. TH with 2.5 g 1⁻¹ PEA is a selective media for B. subtilis alone.MacConkey supplemented with maltose is a selective and differentialculture medium for bacteria designed to selectively isolateGram-negative and enteric bacilli and differentiate them based onmaltose fermentation. Therefore TOP10 ΔmalK mutant makes white colonieson the plates while Nissle makes pink colonies; A is E coli ΔmalK, B isE coli Nissile, C is B subtilis, D is L lactis, E is mixed culture; theimages at MacConkey-/B and E appear pink; the images at MacConkey+/B andE appear pink. FIG. 17 shows selective growth of the bacteria used inthis study on different media and selective plates. It can be seen thatwe clearly, selectively killed the target E coli strain (“E coli” onx-axis in FIG. 17) in the mixed population, whereas the other relatedstrain (“E coli-Nissle”) was not similarly killed. Killing of the targetstrain in the mixed population was 1000-fold in this experiment.

References

-   [1] Zhang, X. Z., & Zhang, Y. H. P. (2011). Simple, fast and    high-efficiency transformation system for directed evolution of    cellulase in Bacillus subtilis. Microbial Biotechnology, 4(1),    98-105. http://doi.org/10.1111/j.1751-7915.2010.00230.x-   [2] Wegmann, U., O'Connell-Motherway, M., Zomer, A., Buist, G.,    Shearman, C., Canchaya, C., . . . Kok, J. (2007). Complete genome    sequence of the prototype lactic acid bacterium Lactococcus lactis    subsp. cremoris MG1363. Journal of Bacteriology, 189(8), 3256-70.    http://doi.org/10.1128/JB.01768-06

TABLE 1 Repeat Sequences of SRBs For Use in the InventionEach of R1 and R1′can be selected from these repeat consensus sequences, eg, whenthe system is an crude oil, natural gas or water recovery, processing or storageequipment. NUMBER SEQ START END OF ID BACTERIUM CRISPR_ID POSITIONPOSITION SPACERS DR (REPEAT) CONSENSUS NO: NOTES DesulphovibrioNC_016803_ 2325998 2326074   1 CCTGGCCTGCCCCAAGTGCAAGG  50desulphuricans 1 ND132 Desulphovibrio NC_016803_ 3491653 3498191  98GTCGCCCCCCACGCGGGGGCGTGGATTG  51 1 desulphuricans 4 AAAC ND132Desulphovibrio NC_005863_ 175898 177711  28 GTCGCCCCCCACGCGGGGGCGTGGATTG125 1 vulgaris subsp. 3 AAAC vulgaris str. Hildenborough DesulphobulbusNC_014972_ 1701541 1707620  92 GTCGCCCCCCACGCGGGGGCGTGGATTG 126 1propionicus DSM 1 AAAC 2032 Desulphovibrio NC_017311_ 170455 172268  28GTCGCCCCCCACGCGGGGGCGTGGATTG 125 1 vulgaris RCH1 1 AAAC DesulphovibrioNC_011883_ 676246 676547   3 TGGAGCGGGAAACGGGATTTGAACCCGC  52desulphuricans 1 subsp. desulphuricans  str. (ATCC 27774) DesulphovibrioNC_011883_ 1083779 1085579  29 GTGTTCCCCACGGGCGTGGGGATGAACC  53desulphuricans 2 G subsp. desulphuricans  str. (ATCC 27774)Desulphovibrio NC_022444_ 430661 430743   1 AACCTTTCTGCAAAAAGGTTTCCCC 54 2 gigas DSM 1382 2 (ATCC 19364) Desulphovibrio NC_022444_ 915564915638   1 CCGCTGGATCCGGCTGCAGCGCC  55 gigas DSM 1382 3 (ATCC 19364)Desulphovibrio NC_022444_ 1994976 1995063   1GTTCACTGCCGCATAGGCAGCTCAGAAA  56 gigas DSM 1382 4 (ATCC 19364)Desulphovibrio NC_022444_ 2555284 2555600   4CACCCGACTATTGAAGTCGGGCCTCATT  57 gigas DSM 1382 5 GAAG (ATCC 19364)Desulphuri- AACCTTTCTGCAAAAAGGTTTCCCC 127 2 spirillum indicum S5Desulphovibrio NC_022579_ 10819 11067   3 GTCAAAACCCATACCGATATGGATACCT 58 hydrothermalis 1 CTTTTGAG Desulphovibrio NC_022579_ 24430 24678   3GTCAAAACCCATACCGATATGGATACCT  59 hydrothermalis 2 CTTTTGAGDesulphovibrio NC_022579_ 36027 36275   3 GTCAAAACCCATACCGATATGGATACCT 60 hydrothermalis 3 CTTTTGAG Desulphovibrio NC_022579_ 118127 118736  8 GTCAAAACCCATACCGATATGGATACCT  61 hydrothermalis 4 CTTTTGAGDesulphovibrio NC_022579_ 2366564 2366737   2CTCAAAAGAGGTATCCATATCGGTATGG  62 hydrothermalis 5 GTTTTGACDesulphovibrio NC_022579_ 2574826 2575933  18GTTCACTGCCGGATAGGCAGCTTAGAAA  63 hydrothermalis 6 DesulphovibrioNC_012796_ 1589785 1591828  30 GTCGCCCCCTGCGCGGGGGCGTGGATTG  64magneticus RS-1 1 AAAC Desulphovibrio NC_012796_ 4725356 4726585  20TTTTCTGAGCTGCCTATGCGGCAGTGAA  65 magneticus RS-1 3 C DesulphovibrioNC_011769_ 241933 242082   1 CATCGACGACGAACCCGGGCACCGCCTG  66vulgaris str. 1 ATGGTCCACGCCGTCATG ‘Miyazaki F’ DesulphovibrioNC_011769_ 2444693 2448088  51 GTCGCCCCTCACGCGGGGGCGTGGATAG  67vulgaris str. 3 AAAC ‘Miyazaki F’ Desulphovibrio NC_008741_ 29677 32622 44 GTTTCAATCCACGCCCCCGCACGGGGGG  68 vulgaris subsp. 1 CGAC vulgaris DP4Desulphuri- NC_014836_ 994780 997087  38 TTTCTGAGCTGCCTATGCGGCAGTGAAC 69 3 spirillum 1 indicum S5 Desulphuri- NC_014836_ 1123444 1127359  54GACCGAAGACCTGTCGGAAACGACGGGG  70 spirillum 2 ATTGAGAC indicum S5Desulphovibrio TTTCTGAGCTGCCTATGCGGCAGTGAAC 128 3 gigas DSM 1382(ATCC 19364) Desulphovibrio NC_022444_ 1994976 1995063   2GTTCACTGCCGCATAGGCAGCTCAGAAA  49 gigas DSM 1382 4 (ATCC 19364)Desulphurivibrio NC_014216_ 1780099 1782570  40CGGTTCATCCCCGCGAGTGCGGGGAACA  71 alkaliphilus  2 T AHT2 DesulphurivibrioNC_014216_ 1785014 1791956 115 TTTCTGAGCTGCCTGTGCGGCAGTGAAC  72alkaliphilus  3 AHT2 Desulphuro- NC_015185_ 267992 268349   5GTTTTATCTGAACGTAGTGGGATATAAA  73 bacterium 1 G thermolithotro- phumDSM 11699 Desulphovibrio NC_007519_ 885036 886223  19CGGTTCATCCCCGCGGGTGCGGGGAACA  74 desulphuricans  1 C G20 Informationfrom CRISPRs Database (www.crispr.u-psud.fr) 1 = Repeat sequence (SEQ IDNOS 51 and 125-126) is common across these bacteria; 2 = Repeat sequence(SEQ ID NOS 54 and 127) is common across these bacteria; 3 = Repeatsequence (SEQ ID NOS 69 and 128) is common across these bacteria.

The entries are read as illustrated by the following example

Desulphovibrio NC_016803_4 3491653 3498191 98GTCGCCCCCCACGCGGGGGCGTGGATTGAAAC 51 1 desulphuricans ND132

A CRISPR array is found in Desulphovibrio desulphuricans ND132 startingat position 3491653 and ending at position 3498191, wherein the arrayhas 98 spacer sequences, each flanked by repeats, where the repeats eachhave the sequence of SEQ ID NOS 51 and 125-126. Such a repeat is alsofound in an array of the other bacteria under note number 1 (last columnin the table).

TABLE 2 SEQ ID NO: SPECIES/STRAIN REPEAT SEQUENCE BACTEROIDES REPEATS107  1. Bacteroides fragilis NCTC 9343GTTGTGATTTGCTTTCAAATTAGTATCTTTGAACCATTGGAAACAGC 2. Bacteroides fragilis 638R 108  3. Bacteroides fragilis NCTC 9343ATTTCAATTCCATAAGGTACAATTAATAC  4. Bacteroides fragilis YCH 46 109 5. Bacteroides helcogenes P 36-108 GTTTCAATCCACACACCCGTATAGGGTGTGAC 110 6. Bacteroides sp CF50 ACTGTTTCTGATATGTCAAAGATAAAATTTTGAAAGCAAATCACAAC111  7. Bacteroides thetaiotaomicron  GAAAAAATACAGTTTCGCTCTCA VP1-5482PREVOTELLA REPEATS 112  8. Prevotella dentalis DSM 3688GTCGCGTCTCACGTAGGCGCGTGGATTGAAAC 113  9. Prevotella denticola F0289ATTGTGCTTGCTACTGCAAAGATACACATTTTGAAGCAATTCACA AC 11410. Prevotella denticola F0289 CTCAATGAGTATCTTCCATTAAAACAAGGATTAAGAC 11511. Prevotella intermedia 17 GTTGTTTTTACCTTGCAAACAGCAGGCAGATACAAC 11612. Prevotella intermedia 17GTTGTATTTGCCAATGCAAAGATACTAATTTTAAAGCTAATCACA AC 11713. Prevotella ruminicola 23 GTTGTATATCATTCCTTTCCTACATCAAACCACAAC

TABLE 3 SEQ PHAGE AR- SPA- ID SOURCE RAY CER SPACER SEQUENCE NO: ICP1_CR1 1a CATTGCAACTATGCAAAATGATGAAGCTAAAA  79 2011_A 2aTGTTAGAGTCGGTAGTATCTGGATGATCGATA  80 3a TTATGTATTGACCCCGACACGCCCCCCGACTG 81 4a TTACAGACGACCTAACTCTTCAGTACCATGAT  82 5aTACATAAGCTGCAACACGGTGTTCGTTTAAGT  83 6a AAAATACGCCTTTTTCCCTTCATCGTTTAAAG 84 7a ACCAACAAATCCCATAAACTGATAACCACGTT  85 8aGTCAACCCTTTGCTTATCTTCCCTATTTAAAT  86 9a TGTTAACCACCGCTTGAAATAATCATGATGCA 87 ICP1_ CR1 1b TGTGTCTATACTCAACCAATTTAAGCGCCGCA  88 2006_E 2bCTACTCTCCCCAATATTAGCCATTCCTAATTC  89 3b GTCACCTTACCGTAAGACAGGCAGTAAAATTA 90 4b AAACTAGTGGACGTAATGCAGTATTCACGGTT  91 CR2 1cATCCACACTACAAATAGAACACTCAACCGTGA  92 ICP1_ CR1 1dTGTGTCTATACTCAACCAATTTAAGCGCCGCA  93 2005_A 2dCTACTCTCCCCAATATTAGCCATTCCTAATTC  94 3d AAACTAGTGGACGTAATGCAGTATTCACGGTT 95 4d ATAATCGTTTTGAGTCTCACCAGCTTTTAGGC  96 CR2 1eATCCACACTACAAATAGAACACTCAACCGTGA  97 2e TATTGATTGGTCTCTAACCTTGGGATGATTAA 98 3e TTCACGGGTAGCAACAGGGTAATAAACCAATA  99 ICP1_ CR1 1fCATTGCAACTATGCAAAATGATGAAGCTAAAA 100 2004_A 2fTGTTAGAGTCGGTAGTATCTGGATGATCGATA 101 3f TAGAAGAGTAATAGGAGCTACTGCAAACTTGT102 4f TAACTATGTGTGGTTTATATTTTGTGTGCAAG 103 5fTTTTGAAACTATTGACAGAAGGTTGGGAACCT 104 6f TTGAGGTTGAACCTCTTCCGGTTCCTCTTCTG105 CR2 1g GTGTATTGCTTGCAGTGGGTTACACACAAGAA 106 Underlined = CRISPRspacers that have 100% identity to sequences within the V. cholerae PLE

TABLE 4 Essential Neutral Homolog Essential homolog in homolog in in InAnnotated V. cholerae V. cholerae E. coli E. coli? function vc0631vc0465 tyrS Yes Tyrosyl tRNA synthetase vc2024 vc0093 plsB Yes Glycerol3 phosphate acyltransferase vc2626 dam No Adenine Methyl- transferasevc2763-vc2767 atpCDGAH No F1 ATP synthase (ε, β, γ, α, δ) subunitsvc2768-vc2770 atpFEB No F0 ATP synthase (B, C, A) subunits

TABLE 5 Gene Feature dnaE DNA polymerase III holoenzyme alpha subunitrecA recombinase A ctxB cholera toxin B mdh malate dehydrogenase gyrBDNA gyrase subunit B tcpA toxin co-regulated pilin A ctxA cholera toxinA subunit rpoA RNA polymerase alpha subunit tcpB toxin co-regulatedpilus biosynthesis protein B asd aspartate-semialdehyde dehydrogenase

TABLE 6 Gene Feature ctxB cholera toxin B tcpA toxin co-regulated pilinA ctxA cholera toxin A subunit tcpB toxin co-regulated pilusbiosynthesis protein B wbet ogawa specific antigen hlyA hemolysin A hapRhemagglutinin/protease regulatory protein rstR cryptic phage ctxphitranscriptional represser mshA mannose-sensitive hemagglutinin A tcpPtoxin co-regulated pilus biosynthesis protein P

TABLE 7 Strains used in Example 9 Strains Description Source E.coli-TOP10 One Shot ® ThermoFischer ThermoFischer TOP10 eletrcompetentcells E. coli-TOP10 malK mutant for differentiation This study ΔmalK onMacConkey agar plates supplemented with maltose. E. coli-Nissle Commonprobiotic also known as Isolated from Mutaflor ® probiotic Mutaflor ®.Bacillus subtilis Supercompetent strain of [1] SCK6 B. subtilis byoverexpression of Xylose inducible ComK. Lactococcus lactis L. lactisMG1363 is the [2] MG1363 international prototype for lactic acidbacteria genetics

TABLE 8 Plasmid used in Example 9 Plasmid Description Source pCasens3Low copy plasmid (~5 copies), This study. spectinomycin resistance andCas9 regulated by a translation theophylline riboswitch. pDual2 Mediumcopy number (~10 copies), This study. chloramphenicol resistance and asgRNA targeting E. coli's genome regulated byt AraC/P_(BAD) expressionsystem.See also Tables 9-11 below, which show sequences used in Example 9.

Sequences:

In an example, one or more spacers of the invention target a respectivesequence in this sequence listing. SEQ ID NOs: 1-44 are Type IICRISPR/Cas system sequences, eg, Streptococcus sequences.

SEQ ID SEQUENCES NO: (ALL 5′ TO 3′) PROMOTER   1 TTGAC   2 TATAATTRANSCRIBED LEADER SEQ   3 TATGAAAA   4 ATTTGAG   5 ATTTGAGG   6 GAG   7GAGG   8 TGAG   9 TGAGG  10 TTGAG  11 TGAGG  12 TTTGAG  13 TTTGAGG  14ATTTGAG  15 AATTTGAG  16 CATTTGAG  17 GATTTGAG  18 TATTTGAG  19CGATTTGAG  20 ACGATTTGAG  21 TCATTTGAG  22 TTCATTTGAG  23 ATCATTTGAG  24TTTCATTTGAG  25 AATCATTTGAG  26 AATTCATTTGAG  27 AAATCATTTGAG  28AAATTCATTTGAG  29 AAAATCATTTGAG  30 AAAATTCATTTGAG REPEAT  31 GTT  32GTTT  33 GTTTT  34 GTTTTT  35 GTTTTTG  36 GTTTTTGT  37 GTTTTTGTA  38GTTTTTGTAC  39 GTTTTTGTACT  40 GTTTTTGTACTC  41 GTTTTTGTACTCT  42GTTTTTGTACTCTC  43 GTTTTTGTACTCTCA  44 GTTTTTGTACTCTCAA  45CAAGGACAGTTATTGATTTTATAATCACTATGTGGGTATAAAAACGT The CRISPR leader inCAAAATTTCATTTGA G the CRISPRI locus of Streptococcus thermophilus strainCNRZI 066  46 AAACAAAGAATTAGCTGATCTTTAATAATAAGGAAATGTTACATTAAThe CRISPR leader in GGTTGGTGGGTTGTTTTTATGGGAAAAAATGCTTTAAGAACAAATGTthe CR1SPR1 locus of ATACTT AGA E. coli W3110 CRISPR system  47MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSK >tr|J7RUA5|J7RUA5_RGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQK STAAU CRISPR-LSEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEK associatedYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQS endonuclease Cas9FIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELR OS = StaphylococcusSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKP aureus subsp. aureusTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENA GN = cas9 PE =3 SV = 1 ELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEV KSKKHPQIIKKG  48MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIG >sp|Q99ZW2|CAS9_SALLFDSGETAE TRP1 CRISPR-ATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEED associated KKHERHPIFGendonuclease NIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLICas9/Csn1 EGDLNPDNSD OS = StreptococcusVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLP pyogenes serotype M1GEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLL GN = cas9 PE = 1 SV =1 AQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLG GD  49[SEQUENCE IS INCORPORATED HEREIN BY REFERENCE FOR   >ENA|HE980450|HEUSE IN THE PRESENT INVENTION] 980450.1 Staphylococcusaureus subsp. aureus ORFX gene and pseudo SCCmec- SCC-SCCCRISPRelement, strain M06/0171 123GCGGATAACAATTACTAGAGAAAGAGGAGAAATACTATTCTTCTCCT pBAV1KTXy1R-CTTTAAATAACGAAAACACCCTGCCATAAAATGACAGGGTGTTGATT short1 CRISPR arrayTCGGCATGAAGCCTTATCTTTGTAGCTTCTGCAAGATTTAAGTAACTGTGTAAGGCGTCCCTTACACTTGCATGTATAGTTATTATACCAGGGGGACAGTGCAATGTCAAGAATAAACTGTAGAATGACTAGTGACTTAAATCTTGAGAGTACAAAAACCCGTTGGAATCGTGATTAATAGTAACTGTTGTTGTACAGTTACTTAAATCTTGAGAGTACAAAAACGGCCGAGAAAAGGAGCTGATTCATAGGACAGTTGTACAGTTACTTAAATCTTGAGAGTACAAAAACTCAAACTTGCCCGTAGTTTATCTTATAGCCGTTGTACAGTTACTTAAATCTTGAGAGTACAAAAACATTTACCTCCTTTGATTTAAG TGAACAAGTTTATCC 124TTAAATCTTGAGAGTACAAAAACCCGTTGGAATCGTGATTAATAGTA Repeat-spacersACTGTTGTTGTACAGTTACTTAAATCTTGAGAGTACAAAAACGGCCG sequenceAGAAAAGGAGCTGATTCATAGGACAGTTGTACAGTTACTTAAATCTTGAGAGTACAAAAACTCAAACTTGCCCGTAGTTTATCTTATAGCCGTTGTACAGTTACTTAAATCTTGAGAGTACAAAAAC 120TTAAATAACGAAAACACCCTGCCATAAAATGACAGGGTGTTGATTTC tracrRNA-encodingGGCATGAAGCCTTATCTTTGTAGCTTCTGCAAGATTTAAGTAACTGTG sequenceTAAGGCGTCCCTTACAC 121 TGTCCTATGAATCAGCTCCTTTTCTCGGCC S1. spacer 1 (DNAPoll 111) [PAM = AAAGAAA, in the target is immediately 3′ of the 3′terminal GCC] 122 GGCTATAAGATAAACTACGGGCAAGTTTGA S2. spacer 2 (tetA)[PAM = TAAGAAA, in the target is immediately 3′ of the 3′ terminal TGA]S thermophilus NNAGAAW Consensus PAM

TABLE 9 Sequence of genetic parts use in pCasens3 (See Example 9)Part name Type DNA sequence 129 Promoterttgacggctagctcagtcctaggtacagtgctagctactagag PJ23100 130 RegulatorAAGTCTAGCGAACCGCACTTAATACGACTCACTATAGGTAC Linker+CGGTGATACCAGCATCGTCTTGATGCCCTTGGCAGCACCC TheophyllineTGCTAAGGTAACAACAAGATG riboswitch 131 RNA guidedATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAAT Cas9 from nucleaseAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCG StreptococcusTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGT pyogensATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAG CTAGGAGGTGACTGA 132Origin of GAGTTATACACAGGGCTGGGATCTATTCTTTTTATCTTTTTTTA pSC101replication and TTCTTTCTTTATTCTATAAATTATAACCACTTGAATATAAACAA repliconAAAAAACACACAAAGGTCTAGCGGAATTTACAGAGGGTCTAGCAGAATTTACAAGTTTTCCAGCAAAGGTCTAGCAGAATTTACAGATACCCACAACTCAAAGGAAAAGGACATGTAATTATCATTGACTAGCCCATCTCAATTGGTATAGTGATTAAAATCACCTAGACCAATTGAGATGTATGTCTGAATTAGTTGTTTTCAAAGCAAATGAACTAGCGATTAGTCGCTATGACTTAACGGAGCATGAAACCAAGCTAATTTTATGCTGTGTGGCACTACTCAACCCCACGATTGAAAACCCTACAAGGAAAGAACGGACGGTATCGTTCACTTATAACCAATACGCTCAGATGATGAACATCAGTAGGGAAAATGCTTATGGTGTATTAGCTAAAGCAACCAGAGAGCTGATGACGAGAACTGTGGAAATCAGGAATCCTTTGGTTAAAGGCTTTGAGATTTTCCAGTGGACAAACTATGCCAAGTTCTCAAGCGAAAAATTAGAATTAGTTTTTAGTGAAGAGATATTGCCTTATCTTTTCCAGTTAAAAAAATTCATAAAATATAATCTGGAACATGTTAAGTCTTTTGAAAACAAATACTCTATGAGGATTTATGAGTGGTTATTAAAAGAACTAACACAAAAGAAAACTCACAAGGCAAATATAGAGATTAGCCTTGATGAATTTAAGTTCATGTTAATGCTTGAAAATAACTACCATGAGTTTAAAAGGCTTAACCAATGGGTTTTGAAACCAATAAGTAAAGATTTAAACACTTACAGCAATATGAAATTGGTGGTTGATAAGCGAGGCCGCCCGACTGATACGTTGATTTTCCAAGTTGAACTAGATAGACAAATGGATCTCGTAACCGAACTTGAGAACAACCAGATAAAAATGAATGGTGACAAAATACCAACAACCATTACATCAGATTCCTACCTACATAACGGACTAAGAAAAACACTACACGATGCTTTAACTGCAAAAATTCAGCTCACCAGTTTTGAGGCAAAATTTTTGAGTGACATGCAAAGTAAGTATGATCTCAATGGTTCGTTCTCATGGCTCACGCAAAAACAACGAACCACACTAGAGA ACATACTGGCTAAATACGGAAGGATCTGA133 Spectinomycin ATGAGGGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCA aadAresistance gene GAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGCATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGCCGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTGAAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGCGCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCG AGATCACCAAGGTAGTCGGCAAA

TABLE 10 Sequence of genetic parts use in pDual2 (See Example 9)Part name Type DNA sequence 134 ArabinoseTTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACC AraC/P_(BAD) expressionGGCACGGAACTCGCTCGGGCTGGCCCCGGTGCATTTTTTAAATA systemCCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGATAGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGCTAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTGGCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATCGGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAGCAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCTGGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAGTAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGATGAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCACCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGAGATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGGGGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCATCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAGCATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAAAGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGATCCTACCTGACGCTTTTTATCGCAACTCTCTACT GTTTCTCCATA 135Chloroamphenicol ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCC catresistance AATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCT geneCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTTCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTTTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGC G 136 Origin ofTTAATAAGATGATCTTCTTGAGATCGTTTTGGTCTGCGCGTAAT p15A replicationCTCTTGCTCTGAAAACGAAAAAACCGCCTTGCAGGGCGGTTTTTCGAAGGTTCTCTGAGCTACCAACTCTTTGAACCGAGGTAACTGGCTTGGAGGAGCGCAGTCACCAAAACTTGTCCTTTCAGTTTAGCCTTAACCGGCGCATGACTTCAAGACTAACTCCTCTAAATCAATTACCAGTGGCTGCTGCCAGTGGTGCTTTTGCATGTCTTTCCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGACTGAACGGGGGGTTCGTGCATACAGTCCAGCTTGGAGCGAACTGCCTACCCGGAACTGAGTGTCAGGCGTGGAATGAGACAAACGCGGCCATAACAGCGGAATGACACCGGTAAACCGAAAGGCAGGAACAGGAGAGCGCACGAGGGAGCCGCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCACTGATTTGAGCGTCAGATTTCGTGATGCTTGTCAGGGGGGCGGAGCCTATGGAAAAACGGCTTTGCCGCGGCCCTCTCACTTCCCTGTTAAGTATCTTCCTGGCATCTTCCAGGAAATCTCCGCCCCGTTCGTAAGCCATTTCCGCTCGCCGCAGTCGAACGACCGAGCGTAGCGAGTCAGTGAGCGA GGAAGCGGAATATATCCCTAGG

TABLE 11 Target sequence (See Example 9) Target sequence and NamePAM (NGG) 137 TAGGTGAAGTCCCTCGCGGATGG rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH

1.-62. (canceled)
 63. A method of modifying a mixed population ofmicrobiota bacteria, the mixed population comprising a first bacterialsub-population and a second bacterial sub-population wherein the firstsub-population comprises a first microbiota species and the secondsub-population comprises a host cell population of a second microbiotaspecies, wherein the second species is a different species than thefirst microbiota species, the method comprising a. combining the mixedpopulation of microbiota bacteria with a plurality of vectors encoding aCas nuclease and host modifying (HM) crRNAs, and b. expressingvector-encoded Cas and HM-crRNAs in host cells,  wherein each HM-crRNAis encoded by a vector engineered nucleic acid sequence and is operablewith vector-encoded Cas in a host cell, wherein the engineered nucleicacid sequence and Cas form a HM-CRISPR/Cas system and the engineerednucleic acid sequence comprises (i) a nucleic acid sequence comprisingspacer and repeat sequences encoding said HM-crRNA; (ii) a nucleic acidsequence encoding a sequence of said HM-crRNA, wherein said HM-crRNAsequence is capable of hybridizing to a host cell target sequence toguide Cas nuclease to the target sequence in the host cell; and optionally the HM-system comprises a tracrRNA sequence or a DNAsequence expressing a tracrRNA sequence;  whereby HM-crRNAs guide Casmodification of host target sequences in host cells, whereby host cellsare killed or the host cell population growth is reduced, therebyreducing the proportion of said host cell population and altering therelative ratio of said sub-populations of bacteria in the mixedbacterial population.
 64. The method of claim 63, wherein the Cas is aCas 3 or
 9. 65. The method of claim 63, wherein the Cas is an E coilCas3 or Salmonella typhimurium Cas3.
 66. The method of claim 63, whereinthe vectors encode a Type I CasA, B, C, D, E and Cas3.
 67. A method ofmodifying a mixed population of microbiota bacteria, the mixedpopulation comprising a first bacterial sub-population and a secondbacterial sub-population wherein the first sub-population comprises afirst microbiota species and the second sub-population comprises a hostcell population of a second microbiota species, wherein the secondspecies is a different species than the first microbiota species, themethod comprising a. combining the mixed population of microbiotabacteria with a plurality of vectors encoding host modifying (HM)crRNAs, and b. expressing HM-crRNAs in host cells,  wherein eachHM-crRNA is encoded by a vector engineered nucleic acid sequence and isoperable with a Cas nuclease in a host cell, wherein the engineerednucleic acid sequence and Cas form a HM-CRISPR/Cas system and theengineered nucleic acid sequence comprises (i) a nucleic acid sequencecomprising spacer and repeat sequences encoding said HM-crRNA; (ii) anucleic acid sequence encoding a sequence of said HM-crRNA, wherein saidHM-crRNA sequence is capable of hybridizing to a host cell targetsequence to guide Cas to the target sequence in the host cell; and optionally the HM-system comprises a tracrRNA sequence or a DNAsequence expressing a tracrRNA sequence;  whereby HM-crRNAs guide Casmodification of host target sequences in host cells, whereby host cellsare killed or the host cell population growth is reduced, therebyreducing the proportion of said host cell population and altering therelative ratio of said sub-populations of bacteria in the mixedbacterial population.
 68. The method of claim 67, wherein the methodreduces host cell population growth by at least 5-fold.
 69. The methodof claim 67, wherein the method inhibits host cell population growth ona surface.
 70. The method of claim 67, wherein the first species has a16s ribosomal RNA-encoding DNA sequence that is at least 80% identicalto an 16s ribosomal RNA-encoding DNA sequence of the host cell species,wherein the growth of the first bacteria in the mixed population is notinhibited by said HM-system.
 71. The method of claim 67, wherein themixed population of step (a) comprises a third bacterial species. 72.The method of claim 67, wherein the mixed population of step (a)comprises a further sub-population of bacterial cells of the samespecies as the host cells, wherein the bacterial cells of said furthersub-population do not comprise said target sequence.
 73. The method ofclaim 67, wherein Cas expression is induced in host cells, whereby saidexpressed Cas and HM-crRNAs are combined in the host cells.
 74. Themethod of claim 67, wherein expression of HM-crRNA from the engineerednucleic acid sequences is inducible in the host cell, the methodcomprising inducing production of HM-crRNAs.
 75. The method of claim 67,wherein the Cas is encoded by the vector.
 76. The method of claim 67,wherein the Cas is encoded by the host cell genome.
 77. The method ofclaim 67, wherein the first species is a gram negative species andoptionally the second species is a gram negative species.
 78. The methodof claim 67, wherein the first species is a gram positive species andthe second species is a gram negative species.
 79. The method of claim67, wherein the mixed population of step (a) comprises a third species.80. The method of claim 79, wherein the third species is a gram negativespecies.
 81. The method of claim 79, wherein the third species is a grampositive species.
 82. The method of claim 81, wherein the first andsecond species are gram negative species.
 83. The method of claim 67,wherein the mixed population of step (a) comprises a furthersub-population of bacterial cells of the same species as the host cells,wherein the bacterial cells of said further sub-population do notcomprise said target sequence.
 84. The method of claim 67, wherein thevectors express single guide RNAs (HM-gRNAs) comprising HM-crRNAsequences.
 85. The method of claim 67, wherein the method comprisesusing endogenous host cell RNase III and/or endogenous host celltracrRNA in the production of HM-cRNAs in the host cells.
 86. The methodof claim 67, wherein said target sequence is conserved in bacteria ofsaid second species.
 87. The method of claim 67, wherein said targetsequence is comprised by an essential gene and/or required for proteinexpression in host cells.
 88. The method of claim 67, wherein the hostscells are of a first strain of said second species and said genetictarget sequence is present in said strain, but the target sequence isabsent in bacteria of the second species which are of a differentstrain.
 89. The method of claim 88, wherein the mixed population of step(a) comprises a sub-population of bacteria of said different strain. 90.The method of claim 89, wherein the first species is a gram negativespecies, and the mixed population of step (a) comprises a sub-populationof bacteria of a third species, wherein the third species is a grampositive species.
 91. The method of claim 89, wherein the first speciesis a gram positive species, and the mixed population of step (a)comprises a sub-population of bacteria of a third species, wherein thethird species is a gram negative species.
 92. The method of claim 90,wherein the second species is a gram negative species.
 93. The method ofclaim 67, wherein the second species is an Enterobacteriaceae species orE coli.
 94. The method of claim 67, wherein the second species is ahuman or animal gut microbiota species.
 95. The method of claim 67,wherein each species is an environmental species, or a human or animalmicrobiota species and/or wherein the host cells are cells of a humanmicrobiota species.
 96. The method of claim 67, for treating orpreventing a disease or condition in a human or animal; or wherein themethod treats or prevents a disease or condition in a human or animal.97. The method of claim 67, wherein the first microbiota species is ahuman gut commensal species and/or a human gut probiotic species. 98.The method of claim 67, wherein the first microbiota species is aBacteroidetes and optionally the host cells are gram positive bacterialcells.
 99. The method of claim 67, wherein the host cell populationconsists of Firmicutes cells.
 100. The method of claim 67, wherein foreach host cell the system comprises components according to (i) to(iv):— (i) at least one nucleic acid sequence encoding a Cas nuclease;(ii) an engineered HM-CRISPR array comprising a spacer sequence andrepeats encoding a HM-crRNA, the HM-crRNA comprising a sequence thathybridises to a host cell target sequence to guide said Cas to thetarget in the host cell to modify the target sequence; (iii) an optionaltracrRNA sequence or a DNA sequence expressing a tracrRNA sequence; (iv)wherein components of the system are comprised by at least one nucleicacid vector that transforms the host cell, whereby the HM-crRNA guidesCas to the target to modify the host target sequence in the host cell;and wherein the target sequence is modified by the Cas whereby the hostcell is killed or host cell growth is reduced; the method comprisingintroducing the vectors of (iv) into host cells and expressing saidHM-crRNA in the host cells, allowing HM-cRNA to hybridise to host celltarget sequences to guide Cas to the targets in the host cells to modifytarget sequences, whereby host cells are killed or host cell growth isreduced, thereby altering the relative ratio of said sub-populations inthe mixed population of bacteria.
 101. The method of claim 100, whereineach vector is a virus or phage.
 102. The method of claim 67, whereineach of the first and second species is a respective gram-positive orFirmicutes species and the growth of the first bacteria is not inhibitedby the HM-system.
 103. The method of claim 67 for treating a host cellinfection of a human or animal subject in a method comprising exposingthe host cells to a first antibiotic simultaneously or sequentially withsaid engineered nucleic acid sequences encoding HM-crRNAs, whereintarget sequences are each comprised by an antibiotic resistance gene forresistance to said first antibiotic, wherein the at least one vector orcells are administered to the subject and the host cell infection istreated in the subject.
 104. The method of claim 67 for treating anindustrial or medical fluid, surface, apparatus or container; or fortreating a waterway, water, a beverage, a foodstuff or a cosmetic,wherein said host cells are comprised by or on the fluid, surface,apparatus, container, waterway, water, beverage, foodstuff or cosmetic,wherein host cells are exposed to said vector(s) or plurality of cellsand host cells growth is inhibited, thereby carrying out said treatment.105. The method of claim 67, wherein each host cell is a Staphylococcus,Streptococcus, Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio,Vibrio or Clostridium cell.
 106. The method of claim 67, wherein eachtarget sequence is comprised by an antibiotic resistance gene, virulencegene or essential gene of the host cell.
 107. The method of claim 67,for increasing the proportion of Bacteroides in the mixed population,wherein said increase is carried out.
 108. The method of claim 107,wherein the proportion of B thetaiotomicron and/or B. fragilis isincreased.
 109. The method of claim 67, wherein the relative ratio ofBacteroidetes versus Firmicutes or gram-positive host cells comprised bythe mixed population is increased.
 110. The method of claim 109, whereincommensal or symbiotic Bacteroidetes in a human or animal are favoured.111. The method of claim 110, further comprising obtaining an amount ofthe bacteria of said mixed population comprising said altered ratio,producing a bacterial culture comprising said obtained bacteria, andadministering the culture to a human or animal thereby favouringcommensal or symbiotic Bacteroidetes in said human or animal.
 112. Themethod of claim 96, further comprising obtaining an amount of thebacteria of said mixed population comprising said altered ratio,producing a bacterial culture comprising said obtained bacteria, andadministering the culture to a human or animal thereby treating orpreventing said disease or condition in said human or animal.
 113. Themethod of claim 67 for Paneth cell stimulation by gut Bacteroides in ahuman or animal, wherein the first bacterial species is a Bacteroidesspecies, wherein the method comprises producing the mixed populationcomprising said altered ratio in said human or animal, and administeringsaid mixed population comprising said altered ratio to the human oranimal, whereby Paneth cells are stimulated.
 114. The method of claim 67for developing an immune response to gut Bacteroides in a human oranimal, wherein the first bacterial species is a Bacteroides species,wherein the method comprises producing the mixed population comprisingsaid altered ratio produced in said human or animal, and administeringsaid mixed population comprising said altered ratio to the human oranimal, whereby said immune response is developed.
 115. A plurality ofbacterial host cells, each cell comprising an engineered nucleic acidvector, wherein the vector encodes a Cas that is expressible in the hostcell, wherein the bacterial host cells are comprised by a mixedpopulation of microbiota bacteria, the mixed population comprising afirst sub-population and a second bacterial sub-population wherein thefirst sub-population comprises a first microbiota species and the secondsub-population comprises a host cell population of a second microbiotaspecies, wherein the second species is a different species than thefirst microbiota species.